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Biochemical and antigenic analysis using monoclonal antibodies of a series of influenza A (H3N2) and (H1N1) virus reassortants
Biochemical and antigenic analysis using monoclonal antibodies of a series of influenza A (H3N2) and (HIN1) virus reassortants J. S. Oxford, T. Corcoran, R. Newman, D. Major and G. C. Schild Reassortant influenza A viruses with high growth capacity in eggs and suitable as candidate vaccine strains or as standard reagents for influenza HA quantification were prepared using the high yielding A/PR/8/34 (HIND as one parent and a number of 'wild' strains of influenza A (HINI) or (H3N2) viruses as the other parent. The genetic and antigenic composition of the reassortants was determined. The parental derivation of genes in the reassortants was established by electrophoretic analysis of virus RNA and virus induced polypeptides. The haemagglutinin (HA) antigens of the three HINI viruses (NIB-6, NIB-7 and NIB-12) were found to resemble those of the parental viruses when tested against a panel of monoclonal antibodies and using the HI test. A similar correspondence between the antigenic characteristics of the HA of the influenza A (H3N2) reassortants (NIB-I, NIB-4, NIB-5, NIB-8 and NIB-I 1) and parental viruses was noted. Therefore laboratory manipulations to produce the reassortants did not result in the selection of significant antigenic variants.
Keywords:Influenza, rcassortants: monoclonal antibodies: polypcptidcs; viral RNA
Introduction Influenza A viruses undergo genetic reassortment at a high frequency when a culture is dually infected with two strains (reviewed in ref. 1). This property has been exploited both for the production of reassortant viruses with high growth capacity in embryonated hen's eggs for the preparation of inactivated influenza vaccines z s and for the development of live, attenuated vaccines ~'-'~. This paper describes some genetic, biological and antigenic characteristics of several reassortant viruses derived from A/PR/8/34 (I-|INI) virus as the high-yielding parent and "wild', low-yielding strains as the other parent. These reassortants have been prepared for experimental use m and as candiate vaccine viruses and also as standards for subsequent qt, antification of the [IA content of inactivated influenza vaccines. The genotype of two similar series of reassortant viruses designated "x' viruses a and NBSA viruses ~ has been published. The genotyping of the NIB viruses described here provides information relevant to the genetic basis of biological properties of the viruses. However, since RNA-containing viruses, including influenza, have a high mutation rate ~2~~ it was important to determine if laboratory manipulation during the production of reassortants could result in the selection of antigenic variants. However, antigenic analysis of Division of Viral Products, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NW3 6RB. (Received 28 January 1985; revised 1 1 June 1985) 0264--410)(/86/010009-06503.00 ((_~1986 Butterworth & Co (Publishers) Ltd.
the haemagglutinin (HA) of the rcassortant or parental viruses with monoclonal antibodies and analysis of the genetic composition of the reassortants has not detected significant laboratory induced changes.
Materials and Methods Viruses High yielding (viz. viruses producing HA yields in eggs of approximately 5000 HA units/ml after 2 days incubation) reassortant viruses designated NIBI-11, were prepared from the high growth parental virus A/ PR/8/34 ( l | l N l ) and the following 'field' isolates: A/ Victoria/3/75 (|13N2) NIB-I: A/England/321/77 (H3N2), NIB-4; A/Texas/I/77 (H3N2), NIB-5: A/ USSR/92/77 (H1N1), NIB-6: A/Brazil/l 1/78 (HIN1), N I B - 7 : A / B a n g k o k / l / 7 9 (H3N2) NIB-8: A/Shanghai/ 31/80 (tt3N2), NIB-I 1. Reassortant viruses were prepared by co-infection of embryonated hen's eggs with two parental viruses. Infective allantoic fluids were harvested at 24 h, incubated with hyperimmune serum prepared against A/PR/8/34 (H IN l) virus and the neutralized virus mixture terminally diluted in eggs. After 48 h incubation, infective allantoic fluid harvests, at the terminal dilutions, were titratcd for HA yield and high HA yielding virus pools were recloned by terminal dilution and characterized genetically and antigenically. Viruses were not further plaque purified. For certain experiments viruses were purified by rate zonal centrifugation in 10-40% sucrose gradients (40 min at 21 000 rev min -j in the SW27 rotor of the Beckmann ultracentrifuge H. Vaccine, Vol. 4, March 1986
9
Analysis of influenza A virus reassortants: d. S. Oxford et al.
Monoclonal and polyclonal antibodies and haemagglutination inhibition (HI) tests
Analysis" of virion RNA by electrophoresis in singledimension polyacrylamide gels
Monoclonal antibodies against the IIA of infucnza (H IN l) and (H3N2) viruses were prepared using standard procedures 15"-~'~. In brief, mice were immunized twice with influenza A virus (at doses of approximately 3000 t l A units virus in the first dose and 50 pg purified virus it] the second dose) and spleen cells removed fl)ur days after the second booster dose of antigen, which was given intravenously. Antibody was prepared as mouse ascitic fuids and used h)r the HI tests after overnight treatment lit 37°C with 4 vol. of r e c e p t o r destroying enzyme (Phillips-Duphar, Holland) to remove any non-specific inhibitors. The micro-HI tests with 9B-well Linbro plates w'i,s used and the antigen challenge dose of virus was carefully standardized by repeat titration to 8 l lA units, hm each experiment the homologous virus was included as a control and the reproducibility of III titrcs was established by experiment. 14I titrcs of less than 1/100 (i.e. less than 5-10% of the homologous HI titrcs) wcrc not found to bc reproducible and were therefore designated as negative. I"or cstimati(m of serological differences between viruses an cightfold or greater difference in HI titrc was considered significant with the monoclonal antibodies.
RNA was extracted from concentrated and purified virus (10 mg ml ~) protein using phenol-SDS tit 560( ` by conventional techniques except that the proteinase K step was ommittcd t*'lx. Electrophoresis of RNA was carried out using 2.6 and 3.0% polyacrylamidc gcls with Locning's buffer. Approximately 10 lag of RNA was applied to each channcl and elcctrophorcsis was carried out at room temperature tit 22 mA for 18 h. After clectrophorcsis, the gel was immersed in 11) vol. of cthidium bromide (4 lag ml ~) tor 10 min at 40( ` and RNA bands werc visualized by fluorescence under u.v. light.
Analysis of influenza A virus-induced polypeptides Vero cells cultured in 24-well tissue culture plates (Linbro FB-24) with wells 2 cm in diameter were infected with allantoic fluid suspensions of influenza virus to give a multiplicity of infection of approximately 1(I p.f.u./cell. After 18 h incubation at 35°( ". in serum and methioninc-frcc Gey's medium, the cells were pulsed h)r 20 min with 2 taCt/well of [~5Slmcthionine (sp. act. approx. I(IM0 Ci mmol l: Amersham International) in 0.2 ml Gey's inedium. The cells were then washed and lyscd with 50 gl/dish of a mixture of 2'",, (v/ v) 2-mcrcaptocthanol it] 0.02 xl Tris--I ICI buffer pl I. (~.8 containing bromophenol blue dye, and heated at l()(P(' for 2 rain. Aliquots (I() lal) were layered onto polyacrylamide slab gels measuring 17 x 33 x (1.1 on1 and having 11.66'7o bisacrvlamide concentration, and electrophorcscd at room temperature for 1(~18 h at 14 mA (constant current). The running buffer contained twice the normal concentration of reagents ~". For comparison of the elcctrophoretic migration rates of polypcptitles of different intlucnza viruses, gels of various concentrations of acrylamide (20%. 15% and 17.5'!',,) wcrc examined.
Virus plaque assays Virus plaque assays were performed in plastic tissue culture plates on monolayers of continuous canine kidney cells (MDCK), grown in Eagles minimal essential medium (MEM, Gibco F-15) I''. The medium was buffered with NaHCO3 and supplemented with 5% fl)etal calf serum. Monolayers were incubated in an atmosphere of 3-5% C()2 and air until confluent (4 days). Viruses as diluted alhmtoic tluids were inoculated and adsorbed liar 3(1~~() rain and monolaycrs were overlaid with a medium comprising MEM, NaltC()~ (I).1769% ), bovine serum albumin (0.14%), D E A E dcxtran (100 lag m l i), TI)CK trypsin (Worthington. Croydon, Surrey, 0.6 units ml i), agar (Oxoid L28, 0.5%) and antibiotics. Plates were incubated for four davs at 33°C and 34°C and stained with naphthalene black. 1"he plaquing characteristics of particular viruses were stable after five passages in vitro at terminal dilution ~" and plaque size and morphology were reproducible in replicate experiments. The plaque morphology of parental viruses was always examined in the same experiment as that of reassortant viruses.
Results
Biochemical analysis of influenza reassortants and parental viruses The gem)types of the reassortant viruses studied, established by comparative electrophoresis of virion RNAs, are shown it] Table 1. In most cases the electrophoretic analysis of virus RNAs gave an unambiguous assignation of parental origin of genes (Figure I). Parental origins of the HA and NA was also determined seroh)gically using conventional Fti. NI ()r
Table 1 Genetic analysis of high growth influenza A reassortant viruses Parental origin of genes Reassortant
Parental Virus
1
2
3
4
5
6
7
8
NIB-1 NIB-4 NIB-5 NIB-6 NIB-7 NIB-8 NIB-11
A/VictoriaJ3/75 (H3N2) A/England/321/77 (H3N2) A/Texas/I/77 (H3N2) A/USSR/92/77 (H1 N1 ) A/Brazil/11/78 (H1 N1 ) A/Bangkok/I/79 (H3N2) A/Shanghai/31/80 (H3N2)
V P P P B P P
V P P U B P S
V P P 9 B P P
V E T U t3 B S
P E P P B B P
V E P U 8 B S
V P P P 9 P P
V E P P B P P
All reassortant viruses were prepared with the high growth influenza A/PR/8/34 (H1 N1 ) virus. Gene designations are (H3N2) viruses genes 1-8 coding for the three polymerase polypeptides PB2, PB1, PA, the haemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M) and non-structural (NSt) polypeptides (reviewed in ref. 20). Note that NIB-7 may represent a high growth clonal variant of the A/Brazil/78 parent rather than a true reassortant virus. The designation of HA and NA was confirmed using HI and NI tests respectively
10
Vaccine, Vol. 4, March 1986
Analysis of influenza A virus reassortants: J. S. Oxford et al.
immuno double diffusion tests to confirm the biochemical data. In addition, for some viruses the origins of certain of the genes could not be assigned unambiguously using the two methods because the RNA gene was not clearly visible on ethidium bromide stained RNA gels (see gene 7 of NIB-7, Figure la) or because no electrophoretic mobility differences were detectable between the corresponding genes of the two parents (see gene 3 of NIB-6, Figure la). In these cases, the genetic composition of the reassortants was established by comparing the electrophoretic mobility of virusinduced polypeptides (data not presented) and the
a,
A
B
C
D
E
F
parental origin of the genc coding for a particular polypeptide thus deduced ."~1. .~.3 . Only an incomplete genotype could be obtained from comparison of clectrophoretic mobility of polypcptides alone, since the PA, PB and PBA polypeptides often co-migrated and could not be readily distinguished.
Biological characterization of reassortants All the reassortants analysed were high yielding (viz. tit least 5000 HA units ml-lin allantoic fluids harvested at 48 h postinfcction) in thcir growth characteristics in embryonated hen's eggs (data not presented). Inspcc-
G
b
A
B
C
Figure 1 Analysis of reassortants of influenza A viruses using get electrophoresis of ssRNA (a) Influenza A (H1N1) virus. Lanes: A/PR8/34; B, A/ Brazil/11/78; C, NIB-7; D, A/PR/8/34; E, NUSSPJ92/77; F, NIB-6; G, NPR/8/34. Genes 1-8 code for PB2, PB1, PA. HA, NP, NA. M and NS1 polypeptides respectively (reviewed in ref. 20). Note that gene 7 is poody resolved for certain of the viruses (channels B, C and E). Gene 4 is also poody resolved (channels B, C, E and F). NIB-7 may represent a high growth clonal variant of the A/Brazil parent rather than a true reassortant virus. The gel illustrated above contained 2.6% polyacrylamide (see Methods). (b) Influenza A (H3N2) virus. Lanes: A, A/-rexas/1/77; B, N IB-5; C, A/PR/8/34 (Hi N1). Note that artefactual band splitting is obtained with gene 7 of AFrexas/1/7? virus. When genes 1-3 were poorly separated the gel was run for a more extended time of 26 h The gel illustrated above contained 2.6% polyacrylamide
Vaccine, Vol. 4, March 1986
11
Analysis of influenza A virus reassortants: d. S. Oxford et al.
tion of the data (Table 1) indicatcd that thc latter characteristic did not co-vary with possession of a particular genc or genc constellation. Similarly. no corrclation was dctectcd bctwecn plaque morphology in MDCK cells or size and gone composition. In particular, in agreement with previous studies in this laboratory 1'; but in contrast to reports of others 23-25, plaque morphology was not exclusively related to a single gent or to the genes coding for I tA or NA. Thus, the morphology and size of plaques induced bv NIB-I and NIB-4 were difl'crent from the respective t~/Victoria/75 and A/England/77 parents, although the rcassortants both derived genes coding for HA and NA from these viruses. NIB-I derived all genes, with the exception of gcnc 5, from thc A/Victoria/75 parent virus. Each reassortant virus had a characteristic plaque morphology which was found stable on serial passage m eggs.
Antigenic analysis of the HA of reassortant viruses The haemagglutinins of the rcassortant and parental viruses were analysed using monoclonal antibodies prepared against the HA of influenza A (II1NI) or (H3N2) viruses. The IIA of viruses NIB-6. NIB-7 and NIB-12 (viruses of t I 1N 1 antigenic subtype) closely resembled the i lA of the corresponding parental virus in antigenic con]position (Tahh, 2) when analysed using a panel of 30 monochmal antibodies prepared against four prototype viruses A/USSR/90/77, A/Brazil/ll/T8. A/Eng/ 333/8(} and A/Baylor/5700/82. Only a single nmnoelonal antibod} (No. 7) reacted to signilicantly higher titres (cightfold) with the virus NIB-7 than with the parental virus.
Table 2
Similarly, for thc intluenza A (t13N2) rcassortants and parcntal viruses analysed a marked similarity in antigenic composition of HA was notcd (Tal~le 3). An exception was the serological reaction of monoclonal antibody 27 which reacted to an eightfold higher III titrc with the NIB-4 reassortant compared to the parental virus. In a numbcr of other instances fourfold antibody titre differences wcrc detected e.g. monoeh~nal antibodies 138, 92. 17, 22, 24, 59, 140 and 52 versus NIB-4 and 33 versus NIB-8. ltowcver, it] previous :~tudics we have not found such fourfold ltl diffcrenccs with monoehmal antibodies to be reproducible or signi-
[icanl 2". The above rcassortant and parental viruses vs.erc also analysed using polychmal antiscra preparcd either by immunization of sheep or goats with purified HA, or from postinfcction ferret antisera. No significant serological differences wcre dctectcd between the tiA of the rcassortant and parental viruses (data not presented).
Discussion Both chined and uncloncd pools of influenza A and B viruses have been shown to contain antigenic wmants in a proportion of approximately 1 in II)5 virions ~~2~. Furthermore, e v e n limited laboratory passage ol influenza viruses can result in the appearance of mutations in all eight virus genes 2~. Recent studies have demonstrated that the host cell used for their cultivation can exert strong selective pressures on influenza viruses, resulting it] the production of antigenic vari-
Serological analysis of influenza A (H1N1) reassortant and parental v,ruses with monoclonal antibodies to HA V,rus used to prepare monoclone
..... A/USSR/92/77
22 70 W18
A~USSR/90/77
2
Monoclonal antibody
NIB-6
A,'Brazil/11,'78
NIB-7
6 400 25 600 6 400
6 400 25 600 6 400
6 400 25 600 1 600
12 800 25 600 3 200
A/Brazil/11.'78
25 25 25 25 25
25 25 25 25 25
25 600 25 600 25 600 25 600 25 600 1600
25 600 25 600 25 600 25 600 25 600 1 600
14 17 23 25 38 44 58 61 85
A/Eng/333/80
-" 12 800 12 800 20O 400 400 1 600 6 400
800 25 600 25 600
1 600 6 400
400 6 400 12 800 40O 3 200 800 1600 3 200 3 200
1 2 7 9 16 26 31 34 43 45 51 69
A/Baylor/5700/82
400 6 400 800
1 600 6 400 400
1 600 25 600 3 200 200
22 29 125 142 170
•)Ht titre -:- 100 Note thai data for NIB-12 is not presented
12
HI fitre versus the following viruses
V a c c i n e , Vol. 4, M a r c h 1 9 8 6
600 600 600 600 600 800
600 600 600 600 600 800
• 6 400 6 400 400 400
400 6 400 800 -
12 3 6 6 . 6
800 200 400 400
6 3 6 6 .
400
400 200 400 400
.
25 6 25 25
600 400 600 600
4OO 1 600 1 600 3 2OO 6 400 12 800
25 6 25 25
600 400 600 600
. 3 200
25 600
12 800
Analysis of influenza A virus reassortants: J. S. Oxford et al. Table 3
Serological analysis of influenza A (H3N2) reassortant and parental viruses with monoclonal antibodies to HA
Vires used to Monoclonal prepare monoclonal antibody antibody A/Vie/3/75 4 10 12 15 17 22 24 27 73 13 59 87 92 138 140 194 35 52 5 9 33 68 125 268
HI titre with the following viruses A/Eng/321/77
NIB-4
A/-fex/1/77
NIB-5
A/Ban/I/79
NIB-8
A/Shang/31/80
-
-
12 800 -
200 1 600 3 200 200 3 200 800 800 1 600
400 3 200 6 400 800 12 800 3 200 6 400 3 200
800 1 600 6 400 3 200 12 800 12 800 3 200 3 200 3 200
800 3 200 6 400 6 400 12 800 12 800 3 200 3 200 3 200
1 600 . 800 200 . . . .
1 600 . 800 200 . . . .
1 600 . . . . .
6 400 12800 12 800 nf
6 400 12800 12 800 nt
3200 1 600 400 3 200 nt
12800 200 6 400 1 600 12 800 nt
6 400 25 600 25 600 25 600 3 200 12 800 12 800
6 400 25 600 25 600 25 600 3 200 25 600 12 800
1 600 1600 . . 1 600 . 3 200
1 600 1600 . . 3 200 . 6 400
-
-
. . 1 600 . 1 600
1 600
A/Bangkok/2/79
nt nt
nt nt
nt 6 400
nt 1 600
. .
. .
. .
. .
NIB-8
nt nt nt nt nt nt
nt nt nt nt nt nt
nt nt nt nt nt nt
nt nl nt nt nt nt
nt nt nt nt nt nt
3 200 3 200 1 600 3 200 1 600 1 600
6 6 6 3 3 3
A/England/23/76
A/Texas/I/77
800 6 400 nt 6 400 12 800 -" 12 800 -
NIB-1 400 6 400 6 400 12 800
. .
. . nt nt nt nt nt nt
.
. . . .
. . .
400 400 400 200 200 200
3 200 3 200 3 200 400 3 200 200
NIB-11
1 600
3 200
3 200 3 200 3 200 200 3 200 100
"HI titres ~<100 nt, not tested
ants > . tlowever, all the inlluenza A ( I l I N I ) and (H3N2) virus rcassortants cxamined in the present study, using a wide range of monoclonal antibodies to I t A . gave serological reactions identical or closely similar to those of the parental viruses. Thus the laboratory manipuhttions to produce the reassortants had not resulted in the emergence of significant antigenic variants. although minor changes could bc detectcd using certain nmnoclonal antibodies. In contrast to our resuits, Downie ~ detected quite marked antigenic differences, using monoclonal antibodies, when six of seven reassortants were c o m p a r e d to parental "wild-type" virus. It is possible that analysis of our reassortants by the sensitive R N A : R N A hvbridisation tcchniquc m'3<3~ would detect further minor changes in H A and indeed in other genes. The significance for the efficacy of inactivated influenza vaccines containing viruses with such minor antigenic changes in the H A is not known. Some persons appcar to have a very limited repertoirc of antibodies to influenza H A and can recognize influenza viruses differing in a single epitopc on the l lA 27. Furthermore "ticld" influenza viruses differentiated by monoelonal antibodies to H A can co-exist and spread in outbreaks of inltuenza in some closed communities >. Therefore it would seem advisable, in the absence of definitive data, to ensure that the H A of any reassortant virus used as a candidatc vaccine strain is as close as possible antigcnieally to the "wild" influenza virus H A , and analysis with monoclonal antibodics is required to establish this relation. The different biological characteristics of influenza virus rcassortants, c o m p a r e d to the parental viruses including virulcncc in nlice 32-34 plaquing TM, sensitivity to amantadinc 3s 37 and high ,yield in eggs 2-~ appear to covary with two or more genes rather than with the cxchangc of a single gene. Morcovcr, the prccisc gcnc
constellation responsiblc for a particular biological function may vary according to the two parental viruses used to produce reassortants. Therefore, for the selection of influenza A viruses for high yield properties for inactivated vaccines or for candidate attenuated vaccine strains as many genes as possible (and preferably six) of the high yielding A/PR/8/34 ( H 1N l ) virus should be transferred to the rcassortant virus. A linal qualification is that such a rcassortant virus possessing only the NA and H A genes of the wild virus may stili not produce t IA yields comparable to A/PR/8/34 (t41N 1) virus, or possess the host range and avirulent characteristics of the parental A/PR/8/34 virus s because of the contribution to virulence and high growth of the two genes 4 and 6 coding for t4A and NA respectively ~.
Acknowledgements We would like to thank R. G. Webster (St Jude Children's Research Hospital, Memphis) and A. Douglas (National Institute for Medical Research, Mill Hill) for kindly supplying a number of the monoclonal antibodies used in the study.
References Burnet, F. M. Portraits of viruses: influenza virus A. Intervirology 1979, 11,201 2 Kilbourne, E. D. Future influenza vaccines and the use of genetic recombinants. Bull. WHO 1969, 41,643 3 Schulman, J. L. Palese. P. Biological properties of influenza A/Hong Kong and PR8 viruses: effects of genes for matrix protein and nucleoprotein on virus yield in embryonated eggs. in: Negative Strand Viruses and the Host Cell (Eds. Mahy. B. W. J. and Barry, R. D ) New York: Academic Press, 1978, pp. 663-674 4 Baez. M., Palese, P. and Kilbourne, E D Gene composition of highyielding influenza vaccine strains obtained by recombination. J. Infect, Dis. 1980. 141,362-365 5 Schulman, J. L Virus-determined differences in the pathogenesis of influenza virus infections, in: Genetics of Influenza Viruses (Eds P. 1
Vaccine, Vol. 4, M a r c h 1986
13
Analysis of influenza A virus reassortants: J. S. Oxford et al.
6 7
8
9 10
11
12
13
14 15
16 17
18
19
20 21
14
Palese and D. W. Kingsbury) Springer-Verlag, Wien, New York. 1983 Beare, A. S and Hall, T. S. Recombinant influenza A viruses as vaccines for man. Lancet 1971,2. 1271 Beare, A S, Schild, G C. and Craig, J W. Trials in man with live recombinants made from A/PR/8/34 (HeN 1) and wild H3N2 influenza viruses. Lancet 1975. 2 729 Oxford, J. S., McGeoch, D. J., Schild, G C. and Beare, A S. Analysis of virion RNA segments and polypeptides of influenza A virus recombinants of defined virulence. Nature 1978. 272, 778 Florent. G Gene constellation of live influenza A vaccines. Arch ViroL 1980, 64. 171 McLaren. C.. Grubbs, G E, Staten. E., Barthlow. W.. Quinnan. G and Ennis F. A Comparative antigenicity and immunogenlcity of A., USSR,'77 influenza vaccines in normal and primed mice. Infect Immun 1980. 28, 171 Downle. J. C. A genetic and monoclonal analysis of h~gh-ylelding reassortants of influenza A virus used for human vaccines J Biol Stand. 1984. 12. 101 Holland, J.. Spindler, K.. Horodyski. F., Grabau, E.. Nichol. S. and VandePol. S. Rapid evolution of RNA genomes. Science 1982. 215. 1577 Yewdell. J. W., Webster. R. G and Gerhard. W. Antigenic variation in three distinct determinants of an influenza type A haemagglutrnin molecule. Nature 1979, 279. 246 Skehel. J J. and Schild, G. C The polypeptide composition of ~nfluenza A viruses. Virology 1971.44. 396 Kohler. G and Milstein, C. Derivatfon of specific antlbody-prOducrng tissue culture and tumor lines by cell fusion. Eur. J. Irnmunol. 1976. 6.511 Laemmh. UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970. 227. 680 Palese, P and Schulman. J. L. Mapping of the ~nfluenza wrus genome: identification of the haemagglutmin and the neuram~n~dase genes Prec. Natl Acad. Sci. USA 1976.73. 2142 Oxford. J S..Klimov. A l . C o r c o r a n . T.Ghendon. Y. ZandSchild. G C B~ochem~cal and serological studies of influenza B wruses compansons of h~storical and recent ~solates V~rus Res. 1984. t, 241 Oxford. J. S. Callow. K A., Corcoran. T and Beare, A S Plaqu~ng characteristics of influenza A virus recombinants of defined genetic composition Arch Virol 1982. 74. 227 Webster. R G. Laver. W. G, A~r. G M and Sch~ld. G C Molecular mechanisms of vanation in influenza viruses. Nature 1982. 296. 115 Oxford. J. S.. Schild. G. C. and Alexandrova. G Electrophoret~c migration rate differences of polypeptides of human influenza A viruses: partial analysis of the genome of ~nfluenza vaccine recombinant wruses. Arch Virol 1980. 65, 277
Vaccine, Vol. 4, March 1986
22 23
24
25 26
27
28
29
30
31
32
33
34
35
36
37
Ritchey, M P. B., Palese, P. and Schulman, J. L. Differences in protein patterns of influenza A viruses. Virology 1977.76, 122 Markoff, L J., Murphy, B R, Kendal, A J. and Chanock, R M Probable association of plaque size with neuraminidase subtype among H3N2 influenza A viruses. Arch. VIroL 1979, 62, 277 Schulman, J. L and Palese, P. Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDCK cells. J Virol. 1977, 24, 170 Almond, J. W. A single gene determines the host range of influenza virus. Nature 1977, 270, 617 Oxford. J. S., Abbo, H., Corcoran, T., Webster. R. G.. Smith. A J. et al. Antigenic and biochemical analysis of field ~solates of influenza B virus: evidence for intra- and inter-epidemic variation. J. Gen. ViroL 1983, 64. 2367 Natah. A., Oxford, J. S and Schild. G C Frequency of naturally occurring antibody to influenza virus antigenic variants selected ~n vitro with monoclonal antibody. J. Hyg. 1981.07. 185 Brand. C. and Palese. P. Sequential passage of influenza vfrus in embryonated eggs or tissue culture: emergence of mutants. Virology 1980, 107. 424 Schild, G. C., Oxford, J S.. de Jong. J C. and Webster. R G Evidence for host-cell selection of influenza virus antigenic variants Nature 1983. 303. 706 Hay. A J. Bellamy. A. R, Abrahams. G. Skehel. J J. Brand, C M and Webster, R. G Procedures for characterization of the genetic material of candidate vaccine strains. Dev. B~ol Stand 39. 15 Klimov. A I and Ghendon. Y. Z Genome analys~s of H1N1 influenza virus strains isolated in the USSR during an epidemic En 1961-62. Arch V~rol. 1981.70, 225 Scholtissek. C.. Vallbracht. A. Flehm~g. B and Rott. R. Correlation of pathogenicity and gene constellation of influenza A viruses II Highly neurovirulent recombinants derived from non-nourowrulent or weakly neurovlrulent parent virus strains Virology 1979. 95. 492 Sugiura. A and Ueda. M Neurovirulence of ~nfluenza wrbs ~n m,ce i Neurovprulence of recombmanls between vfrulent and ~wrulent wrLJ~ strains Virology 1980. 101. 440 Katrak. K S.. Corcoran. T Schlld, G. C and Oxford J S Bioch{.'m,cal relationships of an awrulent mflbenza A virus an• passagt, derived neurowrulent A viruses (manuscript ,n preparation) Lubeck. M. D.. Schulman. J L. and Palese. P Suscept~b,hty of ~nfluenza A viruses to amantadine ,s fnfluenced by the gene coding for M protein J. Virol 1978, 28. 710 Hay. A J. Kennedy. N C T. SkeheI. J.J. and Appleyarc. G The matrix prote~n gene determines amantadme-sens~twrty of ,nfluenza viruses. J. Gen V~rol 1979.42. 189 Scholt~ssek C and Faulkner. G. P Amantadtne resistant and senset~ve influenza A stratus and recomb~nants. J Gen V~rol 1979. 44 8O7
Keywords:Influenza, rcassortants: monoclonal antibodies: polypcptidcs; viral RNA
Introduction Influenza A viruses undergo genetic reassortment at a high frequency when a culture is dually infected with two strains (reviewed in ref. 1). This property has been exploited both for the production of reassortant viruses with high growth capacity in embryonated hen's eggs for the preparation of inactivated influenza vaccines z s and for the development of live, attenuated vaccines ~'-'~. This paper describes some genetic, biological and antigenic characteristics of several reassortant viruses derived from A/PR/8/34 (I-|INI) virus as the high-yielding parent and "wild', low-yielding strains as the other parent. These reassortants have been prepared for experimental use m and as candiate vaccine viruses and also as standards for subsequent qt, antification of the [IA content of inactivated influenza vaccines. The genotype of two similar series of reassortant viruses designated "x' viruses a and NBSA viruses ~ has been published. The genotyping of the NIB viruses described here provides information relevant to the genetic basis of biological properties of the viruses. However, since RNA-containing viruses, including influenza, have a high mutation rate ~2~~ it was important to determine if laboratory manipulation during the production of reassortants could result in the selection of antigenic variants. However, antigenic analysis of Division of Viral Products, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NW3 6RB. (Received 28 January 1985; revised 1 1 June 1985) 0264--410)(/86/010009-06503.00 ((_~1986 Butterworth & Co (Publishers) Ltd.
the haemagglutinin (HA) of the rcassortant or parental viruses with monoclonal antibodies and analysis of the genetic composition of the reassortants has not detected significant laboratory induced changes.
Materials and Methods Viruses High yielding (viz. viruses producing HA yields in eggs of approximately 5000 HA units/ml after 2 days incubation) reassortant viruses designated NIBI-11, were prepared from the high growth parental virus A/ PR/8/34 ( l | l N l ) and the following 'field' isolates: A/ Victoria/3/75 (|13N2) NIB-I: A/England/321/77 (H3N2), NIB-4; A/Texas/I/77 (H3N2), NIB-5: A/ USSR/92/77 (H1N1), NIB-6: A/Brazil/l 1/78 (HIN1), N I B - 7 : A / B a n g k o k / l / 7 9 (H3N2) NIB-8: A/Shanghai/ 31/80 (tt3N2), NIB-I 1. Reassortant viruses were prepared by co-infection of embryonated hen's eggs with two parental viruses. Infective allantoic fluids were harvested at 24 h, incubated with hyperimmune serum prepared against A/PR/8/34 (H IN l) virus and the neutralized virus mixture terminally diluted in eggs. After 48 h incubation, infective allantoic fluid harvests, at the terminal dilutions, were titratcd for HA yield and high HA yielding virus pools were recloned by terminal dilution and characterized genetically and antigenically. Viruses were not further plaque purified. For certain experiments viruses were purified by rate zonal centrifugation in 10-40% sucrose gradients (40 min at 21 000 rev min -j in the SW27 rotor of the Beckmann ultracentrifuge H. Vaccine, Vol. 4, March 1986
9
Analysis of influenza A virus reassortants: d. S. Oxford et al.
Monoclonal and polyclonal antibodies and haemagglutination inhibition (HI) tests
Analysis" of virion RNA by electrophoresis in singledimension polyacrylamide gels
Monoclonal antibodies against the IIA of infucnza (H IN l) and (H3N2) viruses were prepared using standard procedures 15"-~'~. In brief, mice were immunized twice with influenza A virus (at doses of approximately 3000 t l A units virus in the first dose and 50 pg purified virus it] the second dose) and spleen cells removed fl)ur days after the second booster dose of antigen, which was given intravenously. Antibody was prepared as mouse ascitic fuids and used h)r the HI tests after overnight treatment lit 37°C with 4 vol. of r e c e p t o r destroying enzyme (Phillips-Duphar, Holland) to remove any non-specific inhibitors. The micro-HI tests with 9B-well Linbro plates w'i,s used and the antigen challenge dose of virus was carefully standardized by repeat titration to 8 l lA units, hm each experiment the homologous virus was included as a control and the reproducibility of III titrcs was established by experiment. 14I titrcs of less than 1/100 (i.e. less than 5-10% of the homologous HI titrcs) wcrc not found to bc reproducible and were therefore designated as negative. I"or cstimati(m of serological differences between viruses an cightfold or greater difference in HI titrc was considered significant with the monoclonal antibodies.
RNA was extracted from concentrated and purified virus (10 mg ml ~) protein using phenol-SDS tit 560( ` by conventional techniques except that the proteinase K step was ommittcd t*'lx. Electrophoresis of RNA was carried out using 2.6 and 3.0% polyacrylamidc gcls with Locning's buffer. Approximately 10 lag of RNA was applied to each channcl and elcctrophorcsis was carried out at room temperature tit 22 mA for 18 h. After clectrophorcsis, the gel was immersed in 11) vol. of cthidium bromide (4 lag ml ~) tor 10 min at 40( ` and RNA bands werc visualized by fluorescence under u.v. light.
Analysis of influenza A virus-induced polypeptides Vero cells cultured in 24-well tissue culture plates (Linbro FB-24) with wells 2 cm in diameter were infected with allantoic fluid suspensions of influenza virus to give a multiplicity of infection of approximately 1(I p.f.u./cell. After 18 h incubation at 35°( ". in serum and methioninc-frcc Gey's medium, the cells were pulsed h)r 20 min with 2 taCt/well of [~5Slmcthionine (sp. act. approx. I(IM0 Ci mmol l: Amersham International) in 0.2 ml Gey's inedium. The cells were then washed and lyscd with 50 gl/dish of a mixture of 2'",, (v/ v) 2-mcrcaptocthanol it] 0.02 xl Tris--I ICI buffer pl I. (~.8 containing bromophenol blue dye, and heated at l()(P(' for 2 rain. Aliquots (I() lal) were layered onto polyacrylamide slab gels measuring 17 x 33 x (1.1 on1 and having 11.66'7o bisacrvlamide concentration, and electrophorcscd at room temperature for 1(~18 h at 14 mA (constant current). The running buffer contained twice the normal concentration of reagents ~". For comparison of the elcctrophoretic migration rates of polypcptitles of different intlucnza viruses, gels of various concentrations of acrylamide (20%. 15% and 17.5'!',,) wcrc examined.
Virus plaque assays Virus plaque assays were performed in plastic tissue culture plates on monolayers of continuous canine kidney cells (MDCK), grown in Eagles minimal essential medium (MEM, Gibco F-15) I''. The medium was buffered with NaHCO3 and supplemented with 5% fl)etal calf serum. Monolayers were incubated in an atmosphere of 3-5% C()2 and air until confluent (4 days). Viruses as diluted alhmtoic tluids were inoculated and adsorbed liar 3(1~~() rain and monolaycrs were overlaid with a medium comprising MEM, NaltC()~ (I).1769% ), bovine serum albumin (0.14%), D E A E dcxtran (100 lag m l i), TI)CK trypsin (Worthington. Croydon, Surrey, 0.6 units ml i), agar (Oxoid L28, 0.5%) and antibiotics. Plates were incubated for four davs at 33°C and 34°C and stained with naphthalene black. 1"he plaquing characteristics of particular viruses were stable after five passages in vitro at terminal dilution ~" and plaque size and morphology were reproducible in replicate experiments. The plaque morphology of parental viruses was always examined in the same experiment as that of reassortant viruses.
Results
Biochemical analysis of influenza reassortants and parental viruses The gem)types of the reassortant viruses studied, established by comparative electrophoresis of virion RNAs, are shown it] Table 1. In most cases the electrophoretic analysis of virus RNAs gave an unambiguous assignation of parental origin of genes (Figure I). Parental origins of the HA and NA was also determined seroh)gically using conventional Fti. NI ()r
Table 1 Genetic analysis of high growth influenza A reassortant viruses Parental origin of genes Reassortant
Parental Virus
1
2
3
4
5
6
7
8
NIB-1 NIB-4 NIB-5 NIB-6 NIB-7 NIB-8 NIB-11
A/VictoriaJ3/75 (H3N2) A/England/321/77 (H3N2) A/Texas/I/77 (H3N2) A/USSR/92/77 (H1 N1 ) A/Brazil/11/78 (H1 N1 ) A/Bangkok/I/79 (H3N2) A/Shanghai/31/80 (H3N2)
V P P P B P P
V P P U B P S
V P P 9 B P P
V E T U t3 B S
P E P P B B P
V E P U 8 B S
V P P P 9 P P
V E P P B P P
All reassortant viruses were prepared with the high growth influenza A/PR/8/34 (H1 N1 ) virus. Gene designations are (H3N2) viruses genes 1-8 coding for the three polymerase polypeptides PB2, PB1, PA, the haemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M) and non-structural (NSt) polypeptides (reviewed in ref. 20). Note that NIB-7 may represent a high growth clonal variant of the A/Brazil/78 parent rather than a true reassortant virus. The designation of HA and NA was confirmed using HI and NI tests respectively
10
Vaccine, Vol. 4, March 1986
Analysis of influenza A virus reassortants: J. S. Oxford et al.
immuno double diffusion tests to confirm the biochemical data. In addition, for some viruses the origins of certain of the genes could not be assigned unambiguously using the two methods because the RNA gene was not clearly visible on ethidium bromide stained RNA gels (see gene 7 of NIB-7, Figure la) or because no electrophoretic mobility differences were detectable between the corresponding genes of the two parents (see gene 3 of NIB-6, Figure la). In these cases, the genetic composition of the reassortants was established by comparing the electrophoretic mobility of virusinduced polypeptides (data not presented) and the
a,
A
B
C
D
E
F
parental origin of the genc coding for a particular polypeptide thus deduced ."~1. .~.3 . Only an incomplete genotype could be obtained from comparison of clectrophoretic mobility of polypcptides alone, since the PA, PB and PBA polypeptides often co-migrated and could not be readily distinguished.
Biological characterization of reassortants All the reassortants analysed were high yielding (viz. tit least 5000 HA units ml-lin allantoic fluids harvested at 48 h postinfcction) in thcir growth characteristics in embryonated hen's eggs (data not presented). Inspcc-
G
b
A
B
C
Figure 1 Analysis of reassortants of influenza A viruses using get electrophoresis of ssRNA (a) Influenza A (H1N1) virus. Lanes: A/PR8/34; B, A/ Brazil/11/78; C, NIB-7; D, A/PR/8/34; E, NUSSPJ92/77; F, NIB-6; G, NPR/8/34. Genes 1-8 code for PB2, PB1, PA. HA, NP, NA. M and NS1 polypeptides respectively (reviewed in ref. 20). Note that gene 7 is poody resolved for certain of the viruses (channels B, C and E). Gene 4 is also poody resolved (channels B, C, E and F). NIB-7 may represent a high growth clonal variant of the A/Brazil parent rather than a true reassortant virus. The gel illustrated above contained 2.6% polyacrylamide (see Methods). (b) Influenza A (H3N2) virus. Lanes: A, A/-rexas/1/77; B, N IB-5; C, A/PR/8/34 (Hi N1). Note that artefactual band splitting is obtained with gene 7 of AFrexas/1/7? virus. When genes 1-3 were poorly separated the gel was run for a more extended time of 26 h The gel illustrated above contained 2.6% polyacrylamide
Vaccine, Vol. 4, March 1986
11
Analysis of influenza A virus reassortants: d. S. Oxford et al.
tion of the data (Table 1) indicatcd that thc latter characteristic did not co-vary with possession of a particular genc or genc constellation. Similarly. no corrclation was dctectcd bctwecn plaque morphology in MDCK cells or size and gone composition. In particular, in agreement with previous studies in this laboratory 1'; but in contrast to reports of others 23-25, plaque morphology was not exclusively related to a single gent or to the genes coding for I tA or NA. Thus, the morphology and size of plaques induced bv NIB-I and NIB-4 were difl'crent from the respective t~/Victoria/75 and A/England/77 parents, although the rcassortants both derived genes coding for HA and NA from these viruses. NIB-I derived all genes, with the exception of gcnc 5, from thc A/Victoria/75 parent virus. Each reassortant virus had a characteristic plaque morphology which was found stable on serial passage m eggs.
Antigenic analysis of the HA of reassortant viruses The haemagglutinins of the rcassortant and parental viruses were analysed using monoclonal antibodies prepared against the HA of influenza A (II1NI) or (H3N2) viruses. The IIA of viruses NIB-6. NIB-7 and NIB-12 (viruses of t I 1N 1 antigenic subtype) closely resembled the i lA of the corresponding parental virus in antigenic con]position (Tahh, 2) when analysed using a panel of 30 monochmal antibodies prepared against four prototype viruses A/USSR/90/77, A/Brazil/ll/T8. A/Eng/ 333/8(} and A/Baylor/5700/82. Only a single nmnoelonal antibod} (No. 7) reacted to signilicantly higher titres (cightfold) with the virus NIB-7 than with the parental virus.
Table 2
Similarly, for thc intluenza A (t13N2) rcassortants and parcntal viruses analysed a marked similarity in antigenic composition of HA was notcd (Tal~le 3). An exception was the serological reaction of monoclonal antibody 27 which reacted to an eightfold higher III titrc with the NIB-4 reassortant compared to the parental virus. In a numbcr of other instances fourfold antibody titre differences wcrc detected e.g. monoeh~nal antibodies 138, 92. 17, 22, 24, 59, 140 and 52 versus NIB-4 and 33 versus NIB-8. ltowcver, it] previous :~tudics we have not found such fourfold ltl diffcrenccs with monoehmal antibodies to be reproducible or signi-
[icanl 2". The above rcassortant and parental viruses vs.erc also analysed using polychmal antiscra preparcd either by immunization of sheep or goats with purified HA, or from postinfcction ferret antisera. No significant serological differences wcre dctectcd between the tiA of the rcassortant and parental viruses (data not presented).
Discussion Both chined and uncloncd pools of influenza A and B viruses have been shown to contain antigenic wmants in a proportion of approximately 1 in II)5 virions ~~2~. Furthermore, e v e n limited laboratory passage ol influenza viruses can result in the appearance of mutations in all eight virus genes 2~. Recent studies have demonstrated that the host cell used for their cultivation can exert strong selective pressures on influenza viruses, resulting it] the production of antigenic vari-
Serological analysis of influenza A (H1N1) reassortant and parental v,ruses with monoclonal antibodies to HA V,rus used to prepare monoclone
..... A/USSR/92/77
22 70 W18
A~USSR/90/77
2
Monoclonal antibody
NIB-6
A,'Brazil/11,'78
NIB-7
6 400 25 600 6 400
6 400 25 600 6 400
6 400 25 600 1 600
12 800 25 600 3 200
A/Brazil/11.'78
25 25 25 25 25
25 25 25 25 25
25 600 25 600 25 600 25 600 25 600 1600
25 600 25 600 25 600 25 600 25 600 1 600
14 17 23 25 38 44 58 61 85
A/Eng/333/80
-" 12 800 12 800 20O 400 400 1 600 6 400
800 25 600 25 600
1 600 6 400
400 6 400 12 800 40O 3 200 800 1600 3 200 3 200
1 2 7 9 16 26 31 34 43 45 51 69
A/Baylor/5700/82
400 6 400 800
1 600 6 400 400
1 600 25 600 3 200 200
22 29 125 142 170
•)Ht titre -:- 100 Note thai data for NIB-12 is not presented
12
HI fitre versus the following viruses
V a c c i n e , Vol. 4, M a r c h 1 9 8 6
600 600 600 600 600 800
600 600 600 600 600 800
• 6 400 6 400 400 400
400 6 400 800 -
12 3 6 6 . 6
800 200 400 400
6 3 6 6 .
400
400 200 400 400
.
25 6 25 25
600 400 600 600
4OO 1 600 1 600 3 2OO 6 400 12 800
25 6 25 25
600 400 600 600
. 3 200
25 600
12 800
Analysis of influenza A virus reassortants: J. S. Oxford et al. Table 3
Serological analysis of influenza A (H3N2) reassortant and parental viruses with monoclonal antibodies to HA
Vires used to Monoclonal prepare monoclonal antibody antibody A/Vie/3/75 4 10 12 15 17 22 24 27 73 13 59 87 92 138 140 194 35 52 5 9 33 68 125 268
HI titre with the following viruses A/Eng/321/77
NIB-4
A/-fex/1/77
NIB-5
A/Ban/I/79
NIB-8
A/Shang/31/80
-
-
12 800 -
200 1 600 3 200 200 3 200 800 800 1 600
400 3 200 6 400 800 12 800 3 200 6 400 3 200
800 1 600 6 400 3 200 12 800 12 800 3 200 3 200 3 200
800 3 200 6 400 6 400 12 800 12 800 3 200 3 200 3 200
1 600 . 800 200 . . . .
1 600 . 800 200 . . . .
1 600 . . . . .
6 400 12800 12 800 nf
6 400 12800 12 800 nt
3200 1 600 400 3 200 nt
12800 200 6 400 1 600 12 800 nt
6 400 25 600 25 600 25 600 3 200 12 800 12 800
6 400 25 600 25 600 25 600 3 200 25 600 12 800
1 600 1600 . . 1 600 . 3 200
1 600 1600 . . 3 200 . 6 400
-
-
. . 1 600 . 1 600
1 600
A/Bangkok/2/79
nt nt
nt nt
nt 6 400
nt 1 600
. .
. .
. .
. .
NIB-8
nt nt nt nt nt nt
nt nt nt nt nt nt
nt nt nt nt nt nt
nt nl nt nt nt nt
nt nt nt nt nt nt
3 200 3 200 1 600 3 200 1 600 1 600
6 6 6 3 3 3
A/England/23/76
A/Texas/I/77
800 6 400 nt 6 400 12 800 -" 12 800 -
NIB-1 400 6 400 6 400 12 800
. .
. . nt nt nt nt nt nt
.
. . . .
. . .
400 400 400 200 200 200
3 200 3 200 3 200 400 3 200 200
NIB-11
1 600
3 200
3 200 3 200 3 200 200 3 200 100
"HI titres ~<100 nt, not tested
ants > . tlowever, all the inlluenza A ( I l I N I ) and (H3N2) virus rcassortants cxamined in the present study, using a wide range of monoclonal antibodies to I t A . gave serological reactions identical or closely similar to those of the parental viruses. Thus the laboratory manipuhttions to produce the reassortants had not resulted in the emergence of significant antigenic variants. although minor changes could bc detectcd using certain nmnoclonal antibodies. In contrast to our resuits, Downie ~ detected quite marked antigenic differences, using monoclonal antibodies, when six of seven reassortants were c o m p a r e d to parental "wild-type" virus. It is possible that analysis of our reassortants by the sensitive R N A : R N A hvbridisation tcchniquc m'3<3~ would detect further minor changes in H A and indeed in other genes. The significance for the efficacy of inactivated influenza vaccines containing viruses with such minor antigenic changes in the H A is not known. Some persons appcar to have a very limited repertoirc of antibodies to influenza H A and can recognize influenza viruses differing in a single epitopc on the l lA 27. Furthermore "ticld" influenza viruses differentiated by monoelonal antibodies to H A can co-exist and spread in outbreaks of inltuenza in some closed communities >. Therefore it would seem advisable, in the absence of definitive data, to ensure that the H A of any reassortant virus used as a candidatc vaccine strain is as close as possible antigcnieally to the "wild" influenza virus H A , and analysis with monoclonal antibodics is required to establish this relation. The different biological characteristics of influenza virus rcassortants, c o m p a r e d to the parental viruses including virulcncc in nlice 32-34 plaquing TM, sensitivity to amantadinc 3s 37 and high ,yield in eggs 2-~ appear to covary with two or more genes rather than with the cxchangc of a single gene. Morcovcr, the prccisc gcnc
constellation responsiblc for a particular biological function may vary according to the two parental viruses used to produce reassortants. Therefore, for the selection of influenza A viruses for high yield properties for inactivated vaccines or for candidate attenuated vaccine strains as many genes as possible (and preferably six) of the high yielding A/PR/8/34 ( H 1N l ) virus should be transferred to the rcassortant virus. A linal qualification is that such a rcassortant virus possessing only the NA and H A genes of the wild virus may stili not produce t IA yields comparable to A/PR/8/34 (t41N 1) virus, or possess the host range and avirulent characteristics of the parental A/PR/8/34 virus s because of the contribution to virulence and high growth of the two genes 4 and 6 coding for t4A and NA respectively ~.
Acknowledgements We would like to thank R. G. Webster (St Jude Children's Research Hospital, Memphis) and A. Douglas (National Institute for Medical Research, Mill Hill) for kindly supplying a number of the monoclonal antibodies used in the study.
References Burnet, F. M. Portraits of viruses: influenza virus A. Intervirology 1979, 11,201 2 Kilbourne, E. D. Future influenza vaccines and the use of genetic recombinants. Bull. WHO 1969, 41,643 3 Schulman, J. L. Palese. P. Biological properties of influenza A/Hong Kong and PR8 viruses: effects of genes for matrix protein and nucleoprotein on virus yield in embryonated eggs. in: Negative Strand Viruses and the Host Cell (Eds. Mahy. B. W. J. and Barry, R. D ) New York: Academic Press, 1978, pp. 663-674 4 Baez. M., Palese, P. and Kilbourne, E D Gene composition of highyielding influenza vaccine strains obtained by recombination. J. Infect, Dis. 1980. 141,362-365 5 Schulman, J. L Virus-determined differences in the pathogenesis of influenza virus infections, in: Genetics of Influenza Viruses (Eds P. 1
Vaccine, Vol. 4, M a r c h 1986
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Analysis of influenza A virus reassortants: J. S. Oxford et al.
6 7
8
9 10
11
12
13
14 15
16 17
18
19
20 21
14
Palese and D. W. Kingsbury) Springer-Verlag, Wien, New York. 1983 Beare, A. S and Hall, T. S. Recombinant influenza A viruses as vaccines for man. Lancet 1971,2. 1271 Beare, A S, Schild, G C. and Craig, J W. Trials in man with live recombinants made from A/PR/8/34 (HeN 1) and wild H3N2 influenza viruses. Lancet 1975. 2 729 Oxford, J. S., McGeoch, D. J., Schild, G C. and Beare, A S. Analysis of virion RNA segments and polypeptides of influenza A virus recombinants of defined virulence. Nature 1978. 272, 778 Florent. G Gene constellation of live influenza A vaccines. Arch ViroL 1980, 64. 171 McLaren. C.. Grubbs, G E, Staten. E., Barthlow. W.. Quinnan. G and Ennis F. A Comparative antigenicity and immunogenlcity of A., USSR,'77 influenza vaccines in normal and primed mice. Infect Immun 1980. 28, 171 Downle. J. C. A genetic and monoclonal analysis of h~gh-ylelding reassortants of influenza A virus used for human vaccines J Biol Stand. 1984. 12. 101 Holland, J.. Spindler, K.. Horodyski. F., Grabau, E.. Nichol. S. and VandePol. S. Rapid evolution of RNA genomes. Science 1982. 215. 1577 Yewdell. J. W., Webster. R. G and Gerhard. W. Antigenic variation in three distinct determinants of an influenza type A haemagglutrnin molecule. Nature 1979, 279. 246 Skehel. J J. and Schild, G. C The polypeptide composition of ~nfluenza A viruses. Virology 1971.44. 396 Kohler. G and Milstein, C. Derivatfon of specific antlbody-prOducrng tissue culture and tumor lines by cell fusion. Eur. J. Irnmunol. 1976. 6.511 Laemmh. UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970. 227. 680 Palese, P and Schulman. J. L. Mapping of the ~nfluenza wrus genome: identification of the haemagglutmin and the neuram~n~dase genes Prec. Natl Acad. Sci. USA 1976.73. 2142 Oxford. J S..Klimov. A l . C o r c o r a n . T.Ghendon. Y. ZandSchild. G C B~ochem~cal and serological studies of influenza B wruses compansons of h~storical and recent ~solates V~rus Res. 1984. t, 241 Oxford. J. S. Callow. K A., Corcoran. T and Beare, A S Plaqu~ng characteristics of influenza A virus recombinants of defined genetic composition Arch Virol 1982. 74. 227 Webster. R G. Laver. W. G, A~r. G M and Sch~ld. G C Molecular mechanisms of vanation in influenza viruses. Nature 1982. 296. 115 Oxford. J. S.. Schild. G. C. and Alexandrova. G Electrophoret~c migration rate differences of polypeptides of human influenza A viruses: partial analysis of the genome of ~nfluenza vaccine recombinant wruses. Arch Virol 1980. 65, 277
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24
25 26
27
28
29
30
31
32
33
34
35
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Ritchey, M P. B., Palese, P. and Schulman, J. L. Differences in protein patterns of influenza A viruses. Virology 1977.76, 122 Markoff, L J., Murphy, B R, Kendal, A J. and Chanock, R M Probable association of plaque size with neuraminidase subtype among H3N2 influenza A viruses. Arch. VIroL 1979, 62, 277 Schulman, J. L and Palese, P. Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDCK cells. J Virol. 1977, 24, 170 Almond, J. W. A single gene determines the host range of influenza virus. Nature 1977, 270, 617 Oxford. J. S., Abbo, H., Corcoran, T., Webster. R. G.. Smith. A J. et al. Antigenic and biochemical analysis of field ~solates of influenza B virus: evidence for intra- and inter-epidemic variation. J. Gen. ViroL 1983, 64. 2367 Natah. A., Oxford, J. S and Schild. G C Frequency of naturally occurring antibody to influenza virus antigenic variants selected ~n vitro with monoclonal antibody. J. Hyg. 1981.07. 185 Brand. C. and Palese. P. Sequential passage of influenza vfrus in embryonated eggs or tissue culture: emergence of mutants. Virology 1980, 107. 424 Schild, G. C., Oxford, J S.. de Jong. J C. and Webster. R G Evidence for host-cell selection of influenza virus antigenic variants Nature 1983. 303. 706 Hay. A J. Bellamy. A. R, Abrahams. G. Skehel. J J. Brand, C M and Webster, R. G Procedures for characterization of the genetic material of candidate vaccine strains. Dev. B~ol Stand 39. 15 Klimov. A I and Ghendon. Y. Z Genome analys~s of H1N1 influenza virus strains isolated in the USSR during an epidemic En 1961-62. Arch V~rol. 1981.70, 225 Scholtissek. C.. Vallbracht. A. Flehm~g. B and Rott. R. Correlation of pathogenicity and gene constellation of influenza A viruses II Highly neurovirulent recombinants derived from non-nourowrulent or weakly neurovlrulent parent virus strains Virology 1979. 95. 492 Sugiura. A and Ueda. M Neurovirulence of ~nfluenza wrbs ~n m,ce i Neurovprulence of recombmanls between vfrulent and ~wrulent wrLJ~ strains Virology 1980. 101. 440 Katrak. K S.. Corcoran. T Schlld, G. C and Oxford J S Bioch{.'m,cal relationships of an awrulent mflbenza A virus an• passagt, derived neurowrulent A viruses (manuscript ,n preparation) Lubeck. M. D.. Schulman. J L. and Palese. P Suscept~b,hty of ~nfluenza A viruses to amantadine ,s fnfluenced by the gene coding for M protein J. Virol 1978, 28. 710 Hay. A J. Kennedy. N C T. SkeheI. J.J. and Appleyarc. G The matrix prote~n gene determines amantadme-sens~twrty of ,nfluenza viruses. J. Gen V~rol 1979.42. 189 Scholt~ssek C and Faulkner. G. P Amantadtne resistant and senset~ve influenza A stratus and recomb~nants. J Gen V~rol 1979. 44 8O7