the amantadine-sensitivity of recombinant and parental influenza virus strains

Meal Microbiol Immunol (1981) 169:259--268

Medical Microbiology and Immunology �9 Springer-Verlag 1981

The Amantadine-Sensitivity of Recombinant and Parental Influenza Virus Strains

M. Hamzawi*, R. Jennings, and C.W. Potter

Department of Virology, Academic Division of Pathology, University of Sheffield Medical School, Sheffield, S10 2 RX

Abstract. Several wild-type influenza A strains together with recombinants derived from these strains, were tested for sensitivity to amantadine using the in vitro techniques of inhibition in egg-bit culture and plaque reduction in MDCK cells. The results obtained were analysed with reference to the der- ivation of the recombinants. Susceptibility to amantadine was related to the gene coding for matrix protein, and these data are in agreement with previous reports of studies using other series of influenza viruses.

Introduction

Influenza A viruses differ widely in their susceptibility to amantadine (Grunert et al. 1965; Oxford 1977). Recombinant influenza A viruses prepared from amantadine-sensitive and amantadine-resistant parent viruses show segregation of the amantadine-sensitive property independently of the genes coding for the influenza virus haemagglutinin and neuraminidase surface proteins (Tuckova et al. 1973; Appleyard et al. 1977). In recent studies of recombinant influenza A viruses the property of amanradine-sensitivity has been associated with the gene coding for the matrix (M) protein (Lubeck et al. 1978; Hay et al. 1979); however other investiga- tors, using a different series of recombinants, linked amantadine-sensitivity to a combination of genes coding for the nucleoprotein and the neuraminidase antigen, but also suggest that the gene coding for the (M) protein could influence the result (Scholtissek and Faulkner 1979). Thus the gene or gene-constellation determining amantadine-sensitivity may vary for different series of recombinant influenza viruses.

In the present study we have measured the sensitivity to amantandine of a series of genetically-defined influenza A viruses, using growth inhibition in allantois-on-shell cultures and plaque reduction techniques. The findings are related to gene composition of the virus strains, and the contributory role of various genes to amantadine- sensitivity is discussed.

*Corresponding author

0300-8584/0169/0259/$ 02.20

260 M. Hamzawi et al.

Materials and Methods

Viruses

Influenza viruses A/PR/8/34 (HONI), A/Hong Kong/1/68 (H3N2), A/England/42/72 (H3N2), A/Victoria/3/75 (H3N2) and A/USSR/92/77 (H1N1) were kindly supplied by Dr. J.J. Skehel, National Institute for Medical Research, Mill Hill, London. Re- combinant influenza viruses X31 (H3N2) and MRC-2 (H3N2), recombinants of A/PR8 with A/Hong Kong/68 and A/England/72 respectively, were also obtained from Dr. J.J. Skehel. Influenza viruses clone 6 (H3N2), clone 64c (H2N2) and clone 64d (H2N2), all recombinants of A/PR8 and A/England/939/69 (H3N2) viruses, were supplied, together with their parent A/England/939/69 virus, by Dr. A.S. Beare, Common Cold Research Unit, Salisbury. Recombinant influenza A viruses RIT 4228 (H1N1), RIT 4230, recombinants of A/PR/8/34 and A/USSR/77 parents, strains RIT 4204 HIND, a recombinant of A/PR/8/34 and A/Alaska/77 parents, and the recombinant viruses RIT 1454 (H3N2), RIT 1455 (H3N2), RIT 4049 (H3N2) and RIT 4050 (H3N2), recombinants of A/PR/8/34 and A/Victoria/75 parents, were obtained from Dr. G. Florent, Recherche et lndustrie Therapeutique, Rixensaart, Belgium. Influenza A virus RIT 1472 (HON2), a recombinant of A/PR/8/34, A/Victoria/75 parents - was also supplied by Dr. Florent. A group of A/PR8 - A/Victoria/75 recombinant influenza viruses, VP1, VP2, VP4, VP5 and VP7 were prepared in this labora- tory. All bear H3N2 surface antignes.

Preparation and Characterisation of Recombinant Viruses

Recombinant viruses VP1, VP2, VP4, VP5 and VP7 were prepared by mixed infection of MDCK cell monolayers using essentially the methods of Schulman and Palese (1977). Briefly, cell monolayers were co-infected with A/PR/8/34 and A/Victoria/75 viruses at concentrations of 10 and 100 plaque forming units/cell respectively: After adsorption the monolayers were washed, a liquid overlay medium containing 0.25 units/ml of TPCK trypsin (Worthington Biochemical Company, New Jersey, U.S.A.) added, and the infected monolayers incubated at 34~ for 18 h. The culture fluids were then harvested and putative recombinants selected by plaquing the mixed yield in MDCK monlayers in the presence of A/PR8 antiserum. Each isolate was plaque purified at least twice in the presence of A/PR8 antiserum. The resulting isolates were tested by haemagglutination-inhibition (HI) and neuraminidase-inhibition (NI) assays using standard antisera, and virus pools prepared from strains bearing H3N2 antigens.

Putative recombinant viruses were subjected to polypeptide analysis using the methods of Ritchey et al. (1977). Monolayers of MDCK cells were infected with 1 0 - 100 plaque forming units (p.f.u.) of virus and pulsed with 20/aC1 of S 35 methionine (The Radiochemical Centre, Amersham, Bucks.). After 15 rain at RT, the monlayers were washed with cold PBS, lysed with 0.2 ml of PBS containing 2% (w/v) sodium dodeeyl sulphate (SDS), 1.0% (w/v) mercaptoethanol and 6M urea, boiled for two minutes and applied to SDS-polyacrylamine gels. The methods used for polypeptide analysis were those of Laemmli (1970), as modified by Ritehey et al., (1977). After the completion of electrophoresis the gels were removed, covered with cellophane and exposed to X-ray film for 1 -7 days for localisation of the labelled proteins. The

Amantadine-Sensitivity of Influenza Viruses 261

origin of each gene in the recombinants was determined by comparing the migration of the gene product with polypeptides derived from parent viruses tested in the same gel.

Serological Tests

HI tests were carried out by methods described previously (Fenton et al. 1977), using the microtitre technique (Sever 1962). The sera used for identification of the haemagglutinin antigen of recombinant virus strains were monospecific sera against A/PR/8/34 and A/Victoria/75 viruses prepared in ferrets. NI tests were carried out by standard procedures (Aymard-Henry et al. 1973) using antiserum prepared by hyper- immunisation of rabbits with X42 virus (HEqui-lN2).

Determination of Amantadine Sensitivity

a) Egg-bit Assay

A-O-S cultures propared in WHO perspex plates according to the methods of Fazekas de St. Groth (1958), were inoculated with varying concentrations of amantadine and kept at 33~ for two hours. Serial ten-fold dilutions of virus in PBS were then inoculated into the amantadine-treated or untreated A-O-S cultures. Each virus was tested in eight replicate cultures for each concentration of amantadine, and the drug retained in the cultures for the duration of the experiment. After 72 h incubation at 33~ the A-O-S cultures were tested for virus by haemagglutination using 0.5% fowl eryth- rocytes. Virus titres were calculated according to the method of Karber (1931).

b) Plaque Reduction Assay

Virus plaque titrations were carried out in MDCK cell monolayers using the methods of Tobita et al. (1975). Dilutions of virus were tested in both the presence and absence of varying concentrations of amantadine incorporated into the agar overlay. Briefly, virus was adsorbed to MDCK monolayers for 30 min at RT, and the cultures overlaid with Minimal Essential Medium (Gibco Laboratories, Glasgow, Scotland), containing 0.14% (w/v) bovine serum albumin (Armour Pharmaceutical Company, Eastbourne, England), 1% (w/v) Ionagar No. 2 (Difco Laboratories, Detroit, Michigan, U.S.A.), non-essential amino acids, 100/ag/ml DEAE dextran and 0.25 units/ml of TPCK trypsin; to this medium varying concentrations of amantadine were added. After incubation at 33~ in an atmosphere of 5% CO 2 in air for 72 h, the monolayers were stained with 0.1% (w/v) amido black (Hopkins and Williams, Ltd., Essex, England) in 6% glacial acetic acid and 1.36% (w/v) sodium acetate and the plaques counted. The concentration of amantadine giving 50% reduction in plaque numbers in the amantadine-treated monolayers was calculated for each virus tested.


Results

Virus Concentration and Amantadine Sensitivity

The effect of the concentration of influenza A viruses on susceptibility to amantadine was determined in A-O-S cultures treated with 2.5 gtg/ml of amantadine hydrochloride. The results for four influenza viruses are shown in Fig. 1, and are typical of all the results obtained. The relationship between the concentration of virus in the inoculum and sensitivity to 2.5 #g/ml amantadine was linear; however, for influenza viruses A/PR/8/34, A/Okuda/57 and A/Victoria/75, the dose of amantadine used did not inhibit virus replication at the highest concentrations of virus tested.

Fig. 1. Relationship between concentration of A/PR/8/34, A/FM/1/47, A/Okuda/57 and A/Victoria/3/75 influenza viruses and sensitivity to 2.5 gg/ml of amantadine in allantois,n-shell cultures

0

c::

= 50

.L... ES

c . g 50

100

A/PR /

A / O k u ~ 10-2 lO-e

�9 e e

A/V . ~ . lO-a 10-6 10-',o

Log~EBID~/ml

Effect o f Incubation Time on Amantadine Sensitivity

Serial dilutions of influenza A/PR/8/34, A/FM/1/47 or A/Victoria/75 viruses were inoculated into two sets of A-O-S cultures; 24 replicate cultures were used for each dilution. In one set of cultures 2.5 #g/ml of amantadine was included, whilst the other was untreated. Eight replicates of each dilution were tested at each time and, after 1, 2 and 3 days incubation at 33~ the difference in virus infectivity titre for drug- treated and control cultures determined. The results are shown in Fig. 2. The infectivity titre of influenza virus A/PR/8/34 was reduced by 103.2 EBID50/ml following amantadine treatment for 24 h, but no significant inhibition was observed at either 2 or 3 days, post-infection. In contrast, both A/FM/1/47 and A/Victoria/75 influenza viruses showed a marked reduction in titre at 2 and 3 days, but relatively small reduc- tions at one day, post-infection. In consequence of these findings, the inoculation time used for the determination of sensitivity to amantadine in the present study was three days.


~ E

~ < 6.

A/PRS/~ 10.

A/Vict75

24 48 72

Time (hours)

AIFM~ 147

24 48 72

Fig. 2. Relationship between length of incubation and sensitivity of A/PR/8/34, A/FM/1/47 and A/Victoria/3/75 influenza viruses to 2.5 #g/ml of amantadine in allantois-on-shell cultures. o - - o In absence of amantadine, o - - e In presence of 2.5 ~g/ml amantadine

Amantadine Sensitivity of Influenza Viruses Assayed by Plaque Reduction

To confirm the sensitivity tests of influenza A viruses to amantadine assayed by the

A-O-S technique, virus strains VPI, VP2, and VP4, together with their parent viruses,

were tested using the plaque assay. The results are shown in Fig. 3, and it can be seen

that they were in good agreement with those obtained using the A-O-S method. How-

ever, 50% inhibition of plaque formation required higher concentrations of amanta-

dine than 50% inhibition of growth in A-O-S cultures, but the ranking of the results o f the t w o tests was the same.

20

~.~_ 40

g 6o

. . . , , , , , ,

1 5 10 15 20

Amantadine cancentration (I.~/mll

Fig. 3. Sensitivity of recombinant strains VP1, VP2, VP4, and their A/PR/8/34 and A/Victoria/ 75 parent viruses to varying concentrations of amantadine using the plaque reduction assay in MDCK cultures. A--A A/PR/8/34; n--Q A/Victoria/75; o - - o VP1;o- -o VP2; A-zx VP4


Correlation of Gene Constellation with A mantadine Sensitivity

The sensitivity to amantadine of each of the parental viruses used in the present s tudy was determined in A-O-S cultures and the results are shown in Table 1. Of the six parental strains tested only A/PR/8 /34 virus showed resistance to amantadine and the growth of this virus was not inhibited in the presence of 17.5 gtg/ml of the drug. Con- versely, influenza virus A/Hong Kong/68, A/England/69, A/England/72, A/Victor ia / 75, A/USSR/77 and A/Alaska/77 were inhibited in the presence of concentrat ions of

amantadine of < 5.0 pg/ml. The parental origin of the genes was determined for most of the recombinant viruses

used, or else was known from previous reports in the literature, and the origin o f the gene coding for their M protein was correlated with their amantadine-sensitivity as measured in A-O-S cultures. The results are presented in Table 2 and it can be seen that eleven of the nineteen recombinant strains tested derived their M protein from a gene of A/PR/8 /34 origin, and ten of these viruses showed a resistance to amantadine of an order similar to that of their A /PR/8 /34 parent virus. Of eight recombinant viruses whose M protein was coded for by the various wild-type strains, seven were as sensitive to amantadine as these wild-type viruses. No similar degree of correlation with amantadine-sensitivity was found for any other of the genes. Two of the recombinant viruses, RIT 4050 and VPS, showed amantadine-sensitivity intermediate between that of their wild-type parent strain, A/Victoria/75 and A/PR/8/34 , and were inhibited by 10 and 7.5 pg/ml of amantadine respectively. The gene coding for the M protein of RIT 4050 was derived from A/PR/8/34, that coding for the M protein of VP5 from the A/Victoria/75 parent virus; bo th recombinants possessed genes coding for their haemagglutinin, neuraminidase and nucleoprotein antigens derived from A/ Victoria/75 and RIT 4050 also derived its P2 and P3 but not its P1, protein f rom A/Victoria genes. The origin of the P proteins of VP5 has not ye t been determined.

Table 1. Amantadine sensitivity of parental influenza virus strains

Virus Strain Concentration of amantadine (~ug/ml) required for 50% reduction in titre

A/PR/8/34 17.5 A/HK/1/68 < 2.5 A/England/939/69 2.5 A/Eugland/42/72 2.5 AIVictoria/35/75 < 5.0 A/USSR/92/77 2.5 A/Alaska/77 Not tested

Tab

le

2.

Gen

e d

eriv

atio

n

and

am

anta

din

e se

nsi

tiv

ity

o

f re

com

bin

ant

vir

use

s

Rec

om

bin

ant

Gen

e D

eriv

atio

n

Vir

us

Str

ain

P

aren

t V

iru

ses

Pl

P2

P

3

HA

N

P

NA

M

N

S

Co

nce

ntr

atio

n

of

Am

anta

din

e (v

g/m

l)

req

uir

ed

for

50

% r

edu

ctio

n

in t

itre

t2u

r162

6~

Clo

ne

6 A

/PR

8:

A/E

ng

lan

d/6

9

P P

P --

p

- p

p 15

a

Clo

ne

64

c A

/PR

/8:

A/E

ng

lan

d/6

9

- P

P -

P -

P p

20

a

Clo

ne

64

d

A/P

R/8

: A

/En

gla

nd

/69

-

P P

- P

- P

--

> 2

0 a

RIT

4

22

8

A/P

R/8

: A

/US

SR

/77

-

P -

- P

- -

- 2

.5

RIT

4

23

0

A/P

R/8

: A

/US

SR

/77

-

P P

- P

- -

- <

2.5

RIT

4

23

2

A/P

R/8

: A

/US

SR

/77

-

- P

..

..

P

- >

20

a

RIT

4

05

0

A/P

R/8

: A

/Vic

tori

a/7

5

- P

P -

- -

P P

lO b

RIT

4

04

9

A/P

R/8

: A

/Vic

tori

a/7

5

? ?

? .

..

..

<

2.5

RIT

1

45

4

A/P

R/8

: A

/Vic

tori

a/7

5

--

P P

..

..

..

2.

5

RIT

1

45

5

A/P

R/8

: A

/Vic

tori

a/7

5

? ?

? .

..

..

.

2.5

RIT

1

47

2

A/P

R/8

: A

/Vic

tori

a/7

5

? ?

? P

- -

P 3.

5

VP

1

A/P

R/8

: A

/Vic

tori

a/7

5

P P

P --

P

- P

P >

20

a

VP

2

A/P

R/8

: A

/Vic

tori

a/7

5

P P

P --

-

- P

P >

20

a

VP

4

A/P

R/8

: A

/Vic

tori

a/7

5

P P

? .

..

..

5

.0

VP

5

A/P

R/8

: A

/Vic

tori

a/7

5

? ?

? .

..

.

? 7

.5 b

VP

7

A/P

R/8

: A

/Vic

tori

a/7

5

P -

- -

P -

P P

20

a

X3

1

A/P

R/8

: A

/Ho

ng

K

on

g/6

8

P P

P -

P -

P P

17

.5 a

MR

C2

A

/PR

/8:

A/E

ng

lan

d/7

2

P -

P -

- -

P P

17

.5 a

RIT

4

20

4

A/P

R/8

: A

/Ala

ska

/77

P

--

P -

P -

P P

> 2

0 a

2.

O

n t~

P =

gen

e d

eriv

ed

fro

m

A/P

R/8

/34

; ~-

~- g

ene

der

ived

fr

om

w

ild

-ty

pe

par

ent;

?

= de

riva

tion

of

gen

e n

ot

kn

ow

n

a =

aman

tad

ine

resi

stan

t

b =

of

inte

rmed

iate

se

nsi

tiv

ity

to

am

anta

din

e

Dat

a o

n

gen

e d

eriv

atio

n

of

clo

ne

6, c

lon

e 6

4c,

cl

on

e 6

4d

an

d

MR

C-2

fr

om

F

lore

nt

et a

l.,

Arc

hiv

es

of

Vir

o}

og

y

64

:17

1

(19

80

);

on

gen

e de

riva

tion

o

f X

31

fr

om

B

aez

et a

l.,

Jou

rnal

o

f In

fect

iou

s D

isea

ses

14

1:3

62

(1

98

0);

o

n

gen

e d

eriv

atio

n

of

RIT

4

22

8,

RIT

4

23

0,

RIT

4

23

2

and

R

1T

42

04

fr

om

Flo

ren

t,

pel

~o

nal

co

mm

un

icat

ion

(1

97

9)

IO

O~

t.n


Discussion

Although the mechanism of antiviral activity of amantadine remains unclear, the inhibitory effect of this compound on most influenza A viruses can be shown by a variety of in vitro and in vivo methods (Jensen and Liu 1963, Oxford and Schild 1967;Skehel et al. 1977). Not all influenza virus strains are sensitive to amantadine and the genetic basis of sensitivity has been investigated by several groups. Using amantadine-sensitive and amantadine-resistant parent influenza viruses, and study- ing drug-resistance of recombinant strains derived from these viruses in plaque reduction tests, amantadine-sensitivity has been associated with the gene coding for the M protein (Lubeck et al. 1978; Hay et al. 1979). In contrast, other workers, also using the plaque reduction technique, have linked amantadine-sensitivity to the genes coding for the nucleoprotein and neuraminidase antigens (Scholtissek and Faulkner 1979). However, these workers used animal influenza virus strains, and their findings may not necessarily apply to human influenza viruses. In the present studies, amantadine-sensitivity of parental and recombinant viruses has been carried out using growth inhibition in A-O-S cultures, and this technique was found to give similar results to those obtained by plaque reduction.

In the present study, 22 virus strains derived by recombination of influenza virus A/PR/8/34 with six other influenza virus strains were assessed for their sensitivity to amantadine. The results show that influenza virus A/PR/8/34 was drug-resistant, whilst all other parent strains were sensitive. The variability of recombinant influenza viruses in their drug sensitivity allows an analysis of the genetic factor(s) involved, and the present results indicate that sensitivity to amantadine was associated with the gene coding for the M protein. Thus, ten strains deriving their gene for M protein from A/PR/8/34 virus were amantadine-resistant, whilst seven strains which derived this gene from a drug-sensitive wild-type virus were amantadine-sensitive. These results are in accordance with those of other workers (Lubeck et al. 1978; Hay et al. 1979), but care must be taken in their interpretation as the full gene composition of some strains remains unknown.

Recombinant viruses RIT 4050 and VP5 were of intermediate resistance to amantadine and such strains have been reported by others (Lubeck et al. 1978). The existence of these intermediately sensitive strains may reflect an influence of gene products not directly concerned with amantadine-sensitivity, on the M protein. Ex- tragenic suppressor mutations affecting the products of genes bearing temperature- sensitive lesions have been reported in reoviruses (Ramiq and Fields 1979), and may be responsible for the suppression of the temperature-sensitive lesions seen in some influenza virus strains (Massicot et al. 1980). The P1 gene product has been implicated in this respect in influenza virus sensitivity to amantadine (Lubeck et al. 1978).

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Received February 17, 1981

the amantadine-sensitivity of recombinant and parental influenza virus strains

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