73
J. gen. Virol. (I972), x5, 73-77
Printed in Great Britain
Segregation of Antigenic and Biological Characteristics during
Influenza Virus Recombination
(Accepted I6 December I97I)
Genetic recombination between different strains of influenza A virus takes place readily
under laboratory conditions (reviewed by Kilbourne, I963) and this fact has recently found
practical application in techniques for 'tailor making' antigenic 'hybrid' strains of influenza,
the antigenic composition of which is pre-determined by the appropriate choice of parent
strains (Tumova & Pereira, I965 ; Kilbourne et al. I967; Webster, I97O; McCahon & Schild,
I97I). Recombination has also been suggested as a method for the production of strains
with high growth capacity in the embryonated egg for use in the preparation of inactivated
influenza vaccines (Kilbourne & Murphy, I96O) and recently Kilbourne (I969) has reported
the isolation of a recombinant virus (X-30 using AO/PR8/34 virus and A2/HONG KONG/68 as
parent viruses. X-31 was considered as appropriate for vaccine production since it exhibited
the high yielding growth characteristics of AO/VR8 but was antigenically identical to the
current epidemic virus A2/HONG KONG/68.
In this report we describe the segregation by recombination of four properties of the
influenza virus, namely, antigenic characteristics of the envelope proteins (haemagglutinin
and neuraminidase antigens), growth capacity in the embryonated egg and mouse pathogenicity. Such studies form a useful basis for future attempts to use recombination for the
preparation of strains of virus suitable for use in both inactivated and attenuated vaccines.
The recombination system that was used involved the partial inactivation by u.v. light
of one of the parent viruses (Ao parent - high growth capacity in eggs) prior to recombination in the embryonated egg with the live parent virus (A2 p a r e n t - low growth
capacity). In this system the amount of parental viruses in the yield was small and so it
was possible to isolate recombinants without the use of selection pressures, such as specific
antisera, which are normally necessary in other systems (Kilbourne et al. I967; Webster,
I97O) to remove the large excess of parental viruses. In the course of this work a variety of
recombinants were isolated including some, which like X-3I, might be considered as potentially suitable for the production of inactivated virus vaccines.
The parent viruses used in this study were AO/PR8/34 and AZ/ENGLAND/939/69 (kindly
provided by Dr A. S. Beare, MRC Common Cold Research Unit, Harvard Hospital,
Salisbury). AO/PR8/34 was a mouse pathogenic strain which produces high yields of virus
after 48 hr incubation in the allantoic cavity. A2/EN~LAND/939/69 was a recent isolate of the
current epidemic strain A2/HONa KONC/68 which is not pathogenic in mice and which produces low yields of virus in eggs. Both the envelope antigens, haemagglutinin and neuraminidase, of the two parent viruses were distinct so that their identification in recombinant
viruses was readily achieved.
The two viruses were recombined in the embryonated egg and clones were isolated at
limit dilution as illustrated in Fig. I. Prior to recombination the Ao parent virus was diluted
I in 33 and inactivated to I ~ survival of its original infectivity by u.v. light as described by
Tumova & Pereira, I965. This material was then diluted and o.I ml. (IO 7 EID5o) was
inoculated into the allantoic sac of a io-day-old embryonated egg. The egg was then inoculated with o-I ml. of neat allantoic fluid stock of the A2 parent virus 0 o s EID 5o) and incu-
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A 0 / PR8/34
(10 ~ EID50 - partially
inactivated with u.v. l i g h t high g r o w t h potential)
A 2/ENGLAND/939/69
(108 EID50 - live virus low g r o w t h potential)
Allantoic sac of 10 d.o. egg
15 hr at 35°
I
Isolation of clones at limit dilution
AOS cultures
(13 isolates)
10 d.o. eggs
(21 isolates)
I
Preliminary examination
*5
13
Presumptive
Parental
recombinants A 0 virus
3
Parental
A2 virus
I
Cloned by t w o serial limit dilution passages in eggs o r AOS cultures
3 o r more clones of each isolate examined for A 0 and A 2 characteristics
4
9
R e c o m b i n a n t Parental
viruses
A 0 virus
7
Recombinant
viruses
* O n e p r e s u m p t i v e r e c o m b i n a n t p r o v e d t o be a m i x t u r e of t h r e e
distinct r e c o m b i n a n t s on f u r t h e r cloning
Fig. I. Production and isolation of influenza virus recombinants.
bated at 35 ° for I5 hr. The limit dilution technique used both in the isolation and subsequent
cloning of isolates was as follows: the virus preparation was treated in a Burndept Ultrasonic cleaner (input 8o Kcyc./sec.) for 3o sec., diluted in 2-fold or 3-fold steps and then
inoculated into io-day-old embryonated eggs or allantois-on-shell cultures (AOS cultures)
using a minimum of eight replicate cultures for each dilution. These AOS cultures were prepared and incubated according to the method described by Fazekas de St Groth & White,
(i958) except that the medium used was Leibovitz L~ 5 (Eeibovitz, ~963) to which had been
added Ampicillin (250 #g./ml.) and Amphotericin B (25 #g./ml.) and that the plates were
individually sealed with 'Sellotape' and placed in polythene bags after the virus had been
added. The limit dilution clones were isolated from cultures in which the virus dilution used
infected only approximately Io % of the replicates as indicated by positive haemagglutination when tested with fowl erythrocytes. At least three clones of each isolate (usually from
first and second limit dilution steps) were examined for Ao and A2 characteristics in parallel
with limit dilution clones from eggs which received either of the parent viruses (Table I).
Unfortunately the importance of the use of ultrasonic treatment to disrupt virus aggregates
prior to limit dilution isolation was not realized in the early stages of this work and it was
not used in the isolation of some of the original isolates although it was used in the subsequent cloning of such isolates. Egg isolate number 64 was one of these original isolates
for which ultrasonic treatment was not used and therefore aggregation seems the most likely
explanation as to why it gave three distinct recombinants (64b, 64c, and 64d ) on
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Table I. Immunological and biological characteristics of isolates*
Number Antigenic compositiont
of
^
~
Growth capacity
isolates
Neurin io-day-old
examined glutinin
aminidase embryonated eggs§
~
Viruses infecting
Inactivated Ao (IO7 EID 50)
Live A2 (Io 8 EID5o)
6
6
I
I
Inactivated Ao (IO7 EID 50) x
Live A2 (IOs EID5o)
2
3
I
I
I
~9
Ao
A2
A2
A2
A2
A2
Ao
A2
A2
A2
Ao
Ao
A2
Az
A2
Aa
A2
A2
Ao
Ao
Ao
Ao
Mouse
virulence$
High
Low
High
Intermediate
High
Intermediate
High
High
Low
High
High
Virulent
Avirulent
Virulent
Virulent
Avirulent
Avirulent
Virulent
Avirulent
Avirulent
Virulent
Virulent
* These isolates were obtained at limit dilution from the eggs of the experiment described in the text. The
isolates derived from the mixedly infected egg (I& EID5o of inactivated Ao virus+ Io* of live A2 virus)
were cloned twice more at limit dilution in eggs or AOS cultures and at least two clones were examined for
Ao and A2 characteristics.
t This is based on haemagglutination and neuraminidase inhibition tests.
§ High growth capacity = 512o H.A.U./o'25 ml., intermediate = 640 to t28o H.A.U./o.25 ml., low =
r 6oH.A.U,/o'25 ml.
$ Mouse virulent = extensive pulmonary consolidation after intranasal inoculation of Io to I H.A.U.
(Horsfalt, 1939).
Table 2. Recombinants with characteristics of potential vaccine strains
Recombinants
Parental viruses
Strain
designation
6
7
64c
64d
AO/PR 8/34
A2/ENGLAND/
939/69
Antigenic composition
c
~ , - a
HaemagNeurglutinin
anainidase
Az/HK
Az/HK
Az/HK
Az/HK
A2/HK
Az/HK
Az/HK
Az/HK
Ao
Ao
Az/HK
Az/HK
Growth capacity
in Io-day-old
embryonated eggs
(H.A.U./o'25 ml.)
z56o-512o
2560-5120
64o-128o
64o-128o
256o-512o
32-I28
Mouse lung virulence
(lung lesion score)*
2'5-3'5t
o-o-6
2' I--2'2
o-o.6
2'5-3-6
0-0.8
* InocuIum virus was diIuted to contain 25 H.A.U./o'25 mI. Lung lesion scores in Palles mice (5 to 7 weeks
of age) were assessed six days after inoculation using the inoculation method and scoring system described by
Horsfall (I939).
t Range of values in tests with at least three clones of each recombinant or parent virus strain.
subsequent cloning (see Fig. I) a l t h o u g h the possibilities o f a heterozygote a n d / o r r e c o m b i n a tion during isolation c a n n o t be excluded. N o evidence o f aggregation or heterozygosis was
ever seen when ultrasonics were used p r i o r to limit dilution.
Eleven out o f 36 isolates derived f r o m the mixedly infected egg were r e c o m b i n a n t on the
basis o f the four markers examined and eight different types o f r e c o m b i n a n t were found.
T h e markers used were stable since the properties o f all clones derived f r o m any p a r t i c u l a r
cloned isolate or f r o m the two parental viruses, were identical. Th er e was no evidence of
c o v a r i a t i o n between any o f the f o u r m a r k e r s used, suggesting that they reflect different
p r i m a r y gene functions. H o w e v e r , in the case o f g r o w t h capacity in eggs, the occurrence o f
r e c o m b i n a n t s with intermediate g r o w t h capacity suggests that this m a r k e r m a y be dependent on m o r e t h a n one p r im a r y gene function. Since there are t w o possible alternatives
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76
i
l
I
512
256
128
o
64
i
-"Y .,_,°'" -.-......
•
Ir S
I
,111
I
25
I.
t
30
35
Temperature (o)
I
40
Fig. 2. Growth capacity of parent and recombinant viruses at various temperatures in AOS cultures.
AOS cultures (approximately 3 x io 5 cells) were infected with z H.A.U. of virus and incubated with
shaking in water baths at the temperatures shown for 24 hr. The haemagglutinin produced was
measured after the cultures were frozen and thawed and treated with ultrasonic vibration. Each
point represents the average value for two replicate cultures. O
O, Ao parent virus; ~ - - - - O ,
Az parent virus; [] . . . . ½, recombinant virus; • . . . . l , recombinant virus 64d.
(Ao or A2) for each of the four markers then theoretically there are 24 possible arrangements
of these four markers. Two of these arrangements will be represented by the parent viruses
and six of the other possibilities have been obtained (see Table r - for the purpose of this
calculation we have ignored those recombinants with intermediate growth capacity in eggs).
Of the eight remaining theoretically possible recombinants six would have low growth
capacity in eggs and this could explain why they have not been detected in the yield since it
contains principally virus of high growth capacity (32 out of 36 isolates).
Those isolates which had the characteristics of potential vaccine strains were further
cloned and these additional clones were examined to further check the stability of the antigenic and biological characters of these recombinants. All clones of any isolate had the same
properties and the characteristics of the four different types of recombinant vaccine strains
are shown in Table z.
Since the differencebeLween recombinants 6 and 64 c and 7 and 64 d was based on only a
small difference in growth capacity in eggs, this property was studied further by comparing
their growth at various temperatures in AOS cultures with that of the parent viruses (Fig. 2)
and this confirmed that these viruses differed from their parent viruses and each other in this
property. The results for recombinants 6 and 64 c are not shown on the figure since they
closely resembled 7 and 64d respectively.
In some studies when only the type of recombinant with the characteristics of potential
vaccine strains was desired, selection pressure was used to accelerate the process of isolation
of recombinants. A sample of the allantoic fluid from the mixedly infected egg (see Fig. I)
was incubated overnight at 4 ° with I/Ioo rabbit immune serum prepared against Ao/
~'R8/34 and then clones were isolated from this mixture at limit dilution in eggs. This treat-
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77
ment with antibody increased the probability of isolating the required recombinants since
in the absence of antibody treatment 25 out of the 36 isolates (see Fig. I) were parental Ao
virus whereas after antibody treatment all the clones isolated (9 out of 9) possessed A2
haemagglutinin and two clones had the properties of potential vaccine strains.
We conclude from the present studies that the properties of the influenza virus which were
investigated (i.e. haemagglutinin and neuraminidase antigens, growth capacity in eggs and
mouse pathogenicity) can be segregated by recombination and that by the use of the techniques described it is possible to prepare rapidly and reproducibly vaccine strains with high
growth capacity for use against the new epidemic influenza A virus that might arise. In
addition, other recombinants such as antigenic 'hybrids' can be readily isolated.
Recombinant viruses with high growth capacity in eggs will be of value for the preparation
of vaccines containing inactivated virus particles or for possible future vaccines containing
isolated virus subunits. In addition it seems worthwhile to investigate whether recombination
can also be applied to the production of attenuated live virus vaccine strains specially since
the present methods of attenuation are both unpredictable and unreliable (Tyrrell & Beare,
I969). However, little is known of the genetic determinants of virulence and growth characteristics in the influenza virus and their transference during recombination. A recombination
system such as that described in the present study provides the basis for a systematic
approach to this problem and studies to investigate the value of recombination in the preparation of attenuated influenza vaccine strains are currently in progress. (A. S. Beare, personal communication).
We would like to thank Drs A. S. Beare and D. A. J. Tyrrell for helpful discussion during
the course of this work. We would also like to thank Mr B. Precious, Mr V. Law and Mrs S.
Piddington for technical assistance.
Division of Virology
National Institute of Medical Research
Mill Hill, London NW7 1AA
D. MCCAHON
G . C . SCHILD
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(Received I3 October I971)
6
VIR I5
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