Rev. sci. tech. Off. int. Epiz.,
1989, 8 (3), 771-778.
Strain differentiation of foot and mouth disease
virus type Asia 1 isolates of Indian origin
L.M. BELWAL, V.A. SRINIVASAN and RAMA KANT *
Summary: Antigenic variation among aphthovirus type Asia 1 isolates recovered
from India was investigated by a two-dimensional microneutralisation test. Two
vaccine strains and three field strains were employed for reference. Considerable
antigenic variation was observed among the strains. While vaccine strain Asia
1 IND 8/79 exhibited a narrow spectrum, most strains could be related to Asia
1 63/72. A broad spectrum was observed for the strain Asia 1 WBN 117/85,
which has since been recommended for incorporation in the quadrivalent vaccine.
KEYWORDS: Antigenic variation - Aphthovirus - Foot and mouth disease India.
INTRODUCTION
Foot and m o u t h disease (FMD) is endemic in India. Of the seven serotypes of
virus, four (O, A , C, and Asia 1) are prevalent in the subcontinent (1). While type
O accounts for most outbreaks (50-60%), type Asia 1 takes second place with 15-20%,
followed by type A (10-15%) and type C (1-5%). While no significant antigenic
variation has been encountered in type C isolates of Indian origin (unpublished
findings), types O and A have been shown to exhibit moderate to significant antigenic
variation ( l l , 4). Some of these have also been blamed for b r e a k d o w n of vaccine
immunity (5).
Evidence of antigenic variation in Indian strains of serotype Asia 1 was presented
by Rai (6) who further reported the characterisation of a new subtype, Asia 1/2 (7)
on the basis of complement fixation tests.
The present study was aimed at further elucidation of the antigenic variation among
type Asia 1 strains of Indian origin, as well as at selection of an appropriate vaccine
strain.
MATERIALS A N D METHODS
Virus strains
The vaccine strain Asia 1 India 8/79 was obtained from Wellcome F M D
Laboratory, Pirbright, UK, as a suspension of baby hamster kidney (BHK 21 C l . 13)
* Indian Immunologicals, 11-4-657 Lakdi-ka-pul, Hyderabad 500 004, India.
772
cells. Ten strains were obtained from the World Reference L a b o r a t o r y for F M D at
Pirbright as primary bovine kidney, primary bovine thyroid or porcine kidney (IBRS-2)
cell culture isolates. Two strains (Asia 1 H A H 17/86 and H A H 19/86) were obtained
in cattle tongue epithelium from H a r y a n a Agricultural University, Hissar. The vaccine
strain Asia 1 63/72 was obtained from Indian Veterinary Research Institute,
Bangalore. The remaining twelve strains were isolated in our laboratory at Hyderabad.
All the isolates were serially passaged in BHK cell lines until fully adapted. Details
of virus isolates included in the study and their passage histories are given in Table I.
Reference viruses and antisera
Antisera prepared against vaccine strains Asia 1 India 8/79 and Asia 1 63/72 ,
a n d against field strains Asia 1 India 11/80, Asia 1 R A B 64/85 and Asia 1 W B N
117/85 were employed for reference. Anti-140S guinea pig serum against all the strains
was prepared as described by Rweyemamu et al. (10). Briefly, 140S antigen inactivated
with BEI (binary-ethyleneimine) and purified by sucrose density gradient was
emulsified with Freund's incomplete adjuvant and inoculated intramuscularly into
the hind leg of guinea pigs. A booster dose was administered 21 days later. Ten days
after the second dose, the guinea pigs were bled and their serum pooled before use.
Serum was obtained from cattle vaccinated with the vaccine viruses Asia 1 I N D
8/79 and Asia 1 63/72. After preliminary analysis with anti-140S guinea pig antisera,
strain Asia 1 W B N 117/85 was selected as the vaccine strain, and serum was obtained
from cattle vaccinated with it. A group of four to six seronegative steers maintained
FMD-free at the Holding F a r m of Indian Immunologicals was vaccinated with
appropriate monovalent vaccine, repeated after 21 days. The animals were bled on
the 35th day and their serum was pooled before use.
Serum neutralisation test
T h e strains were compared by cross neutralisation in the two-dimensional
microneutralisation test described by Rweyemamu et al. (10). Antigenic diversity was
determined in terms of ' r ' values obtained as follows:
r
_ serum titre against heterologous virus
serum titre against homologous virus
The 'r' values were calculated from mean titres obtained from three replicate tests.
T h e statistical significance of ' r ' values was tested by using an estimated pooled
variance of 0.106 (9). The strains were differentiated at a 9 9 % significance level, which
requires a critical ' r ' value > 0.24 for a strain to be declared homologous in a three
replicate test.
RESULTS A N D DISCUSSION
T h e antigenic relationships amon g Indian isolates of type Asia 1 F M D virus,
expressed as ' r ' values, are presented in Table II for anti-140S guinea pig serum and
in Table III for serum from vaccinated cattle.
3 3 3 3 3 3 3
CS
CC
¡ - U- !_
ca ca ca
Cî3
—
ON T f C^ CN un un un CN NO
oo oo
oo oo
oo oo OO CO OO
oo — un T f m — T}- co r-~ cN T Í - un —
CN
z z z g
O
ca
ca
ca
ca
aioCaCXX
Q Q Q ^
é ü O
cd
ca
ND '—' NO
ca
Tf
ca
CQ
ca 'ca
ovovocn
ND T f
<a
ND ND
OO OO
—• —•
o\
un
oo
—
¡~. —, X X
ca
BK2E/TB6S4B1
B1E/TB8
E/TBK3B3
BK2E/TB7
BT1B1E/TB7
ih BT1E/TB7
>h BT1B/TB5
BK1E/TB5
E/TBT2B5
BT1E/TB4
RT1F./TB3
IIL
WRL
IIL
HAU,
HAU,
BK2E/TB6
E/TBK1B3
B2E/TB8
BK2E/TB6'
BK2E/TB6
IIL
IIL
WRL
WRL
WRL
WRI.
issar
issar
Used as
Candida te strain
Used as
Vaccine strain
Vaccine strain
Status
Oí
CU
I-.
I I
CU CU
-O
ca c a
T3
ca
c
ca
3 3
< < H
ca
H i4
ca
ca
i¿
cu
ON CN
un
N T N O Û T T I C J
oo oo oo oo oo oo oo
oo
r- co CN NO — O O oo
TT T t r m
—< un oo NO
Z Z Q Q Q Q
S S c¿tzc¿XX
TO TO TO TO TO o
TO TO
TO ro ca c a ca c a c a ca ro
ro TO TO coro
TOc oroc oTO
TO CO
roCO CO CO CO CO
W 7 ) ( / ) 7 1 l / l l / l l / l l 7 1 1 / l ( / )
Í/1 W C/1 (/) 1/1
co co
< < < < < < < < < < < < < < < < < < < < <£<<<<<
* Strains identified w i t h prefix I N D obtained from World Réf. Lab., Pirbright, U . K . (e.g. Asia 1 I N D 8/79). Strains identified with prefix other than
I N D isolated and adapted at I I L , Hyderabad (except the strain Asia 1 63/72 which was obtained from Indian Vet. Res. Institute, Bangalore).
** BT = Bovine thyroid cell c u l t u r e ; BK = Bovine kidney cell c u l t u r e ; RS = Porcine kidney cell culture; B = Baby hamster kidney cell line (BHK 21
C I . 13); E/T = ether treated; S = BHK suspension cell c u l t u r e ; W R L = World Ref. Lab. for FMD, Pirbright, U.K.; I V R I = Indian Vet. Res. Institute;
HAU = Haryana Agri. U n i v . , Hissar; I I L = Indian Immunologicals Laboratory, Hyderabad.
dhra Pi
dhra Pi
nil Nac
nil Nac
'nataka
ala
st Bengal
st Bengal
;am
lam
ssa
asthan
asthan
asthan
ryana
ryana
ca c a
S— 1_
d d
Nalgonda
Nizamabad
Coimbatore
Nilgiris
Bellari
Mallanuram
ca
1ca
ca
s—
Nadia
Murshidabad
Dibrugarh
Kamrup
Sambalpur
OOOO
Bikaner
Boondi
Jaipur
Hissar
Hissar
IIL
IVRI
IIL
IIL
d |
i
larat
harashl
harashl
harashl
Source
C¿
c+^
CU
Southern Indi
*
j
Oi Di
RS1E/TB3S5B2
RS1E/TB5
B1E/TB6
BK/1E/TB7
RS2E/TB6
BT/IBK2E/TB6
BK3E/TB5
B7S4B2
BK2E/TB7
BK1E/TB6
Passage
history at the
time of use **
ij
Easter n India
Sta
o
Ahmedabad
Ahmedabad
Ahmedabad
Baroda
Kaira
Kaira
Kheda
Pune
Ahmednagar
Bombay
District
Place <
CD
j j
Northern Indi
Strain
identification
c
Westei m India
Regior
Details of virus strains used in the study
TABLE I
773
774
T A B L E II
'r' values obtained
using anti-140S guinea pig
antisera
Antisera
Viruses
Gujarat
Asia
Asia
Asia
Asia
Asia
Asia
Asia
Asia 1
IND
8/79
Asia 1
63/72
Asia 1
WBN
117/85
Asia 1
IND
11/80
Asia 1
RAB
64/85
IND
IND
IND
GUK
GUB
GUA
GUK
8/79
11/79
3/79
61/84
104/85
25/86
14/87
1.00
0.91
0.87
0.12*
0.25
0.13*
0.20*
0.02*
0.06*
0.13*
0.13*
0.32
0.30
0.14*
0.09*
0.34
0.36
0.44
0.89
0.58
0.27
0.17*
0.26
0.23*
0.19*
0.89
0.26
0.18*
0.04*
0.06*
0.09*
0.04*
0.02*
0.08*
0.02*
Maharashtra
Asia 1
Asia 1 MAA
Asia 1 MAB
63/72
7/85
142/85
0.01*
0.18*
0.14*
1.00
0.48
0.25
0.59
0.87
0.79
0.20*
0.27
0.36
0.13*
0.05*
0.05*
Rajasthan
Asia 1 RAB
Asia 1 IND
Asia 1 RAJ
64/85
45/82
1/86
0.04*
0.03*
0.02*
0.30
>1.00
0.51
0.17*
0.79
0.46
0.26
0.26
0.23*
West Bengal
Asia 1 WBN
Asia 1 IND
117/85
68/79
0.25
0.02*
0.46
0.79
1.00
0.46
0.35
0.08*
0.07*
0.63
Assam
Asia 1 IND
Asia 1 IND
47/82
177/85
0.02*
0.14*
1.00
0.51
0.54
>1.00
0.26
0.42
0.56
0.07*
Haryana
Asia 1 HAH
Asia 1 HAH
17/86
19/86
0.03*
0.17*
0.48
0.50
0.36
>1.00
0.17*
0.19*
>1.00
0.07*
Andhra Pradesh
Asia 1 APN
Asia 1 APN
32/83
6/87
0.03*
0.14*
0.63
0.04*
0.48
0.06*
0.89
0.19*
0.07*
0.02*
Tamil Nadu
Asia 1 IND
Asia 1 IND
11/80
50/80
0.02*
0.02*
0.16*
0.24
0.09*
0.25
1.00
0.15*
0.18*
0.14*
Karnataka
Asia 1 IND
80/79
0.03*
0.66
0.65
0.23*
0.73
Kerala
Asia 1 IND
48/79
0.10*
0.76
0.56
0.14*
>1.00
Orissa
Asia 1 ORS
8/87
0.53
0.12*
0.38
0.32
1
1
1
1
1
1
1
* 'r' significantly less than 1 at p = .01
1.00
>1.00
>1.00
0.03*
775
TABLE
III
Serological interrelationship among type Asia 1 isolates of Indian origin
(Based o n ' r ' values obtained with serum from vaccinated cattle)
Antisera
Viruses
WESTERN INDIA
Gujarat
Ahmedabad
Ahmedabad
Ahmedabad
Baroda
Kaira
Kaira
Kheda
Maharashtra
Ahmednagar
Pune
Bombay
NORTHERN INDIA
Rajasthan
Bikaner
Boondi
Jaipur
Haryana
Hissar
Hissar
EASTERN INDIA
West Bengal
Nadia
Murshidabad
Assam
Dibrugarh
Kamrup
Orissa
Sambalpur
SOUTHERN INDIA
Andhra Pradesh
Nalgonda
Nizamabad
Tamil Nadu
Coimbatore
Nilgiri
Karnataka
Bellari
Kerala
Malapuram
Asia 1
IND 8/79
Asia 1
63/72
Asia 1
WBN 117/85
IND
IND
IND
GUB
GUK
GUK
GUK
8/79
11/79
25/86
104/85
3/79
61/84
14/87
1.00
0.26
0.29
0.11*
0.30
0.30
0.13*
0.19*
0.32
0.25
0.25
0.69
0.12*
0.10*
0.46
0.50
0.68
0.98
0.69
0.81
0.58
MAA
MAB
7/85
63/72
142/85
0.21*
0.13*
0.13*
0.17*
1.00
0.10*
>1.00
0.23*
0.51
RAB
IND
RAJ
64/85
45/82
1/86
0.17*
0.08*
0.08*
0.36
0.23*
0.13*
0.60
0.58
0.39
HAH
HAH
17/86
19/86
0.09*
0.19*
0.20*
0.39
0.89
0.95
WBN
IND
117/85
48/79
0.23*
0.07*
0.17*
0.26
1.00
0.56
IND
ASK
47/82
177/85
0.03*
0.28
0.19*
0.41
0.37
>1.00
ORS
8/87
0.23*
0.25
0.77
APN
APN
32/83
6/87
0.09*
0.09*
0.21*
0.10*
0.45
0.23*
IND
IND
11/80
50/80
0.35
0.05*
0.15*
0.13*
0.42
0.21*
IND
80/79
0.11*
0.26
0.42
IND
48/79
0.20*
0.33
>1.00
* 'r' significantly less than 1 at p =
0.01
776
The vaccine strain Asia 1 I N D 8/79 exhibited a narrow spectrum with respect
t o V values obtained with anti-140S guinea pig serum. However, contemporary
isolates from Gujarat, the state of origin of this strain, appeared homologous to Asia
1 I N D 8/79, as did the strains Asia 1 W B N 117/85 from West Bengal and Asia 1
ORS 8/87. The Gujarat strains isolated after 1979, and those from other states, showed
divergence from the vaccine strain Asia 1 I N D 8/79 when guinea pig serum was used.
The antigenic spectrum of Asia 1 I N D 8/79 obtained with serum from vaccinated
cattle was wider t h a n that of anti-140S serum. Strains from Assam (Asia 1 ASK
177/85) and Tamil N a d u (Asia 1 I N D 11/80) showed closer relationships to Asia 1
I N D 8/79.
A broad antigenic spectrum was exhibited by the vaccine strain Asia 1 63/72 when
anti-140S sera were employed. The 1979, 1984, 1987 Gujarat isolates and one isolate
each from A n d h r a Pradesh, Tamil N a d u and Orissa appeared to be divergent.
However, while higher ' r ' values were obtained against divergent Gujarat isolates,
the overall spectrum with serum from vaccinated cattle was narrower t h a n that with
anti-140S guinea pig serum.
On the basis of 'r' values from anti-140S antiserum and bovine serum, most of
the strains were related to Asia 1 W B N 117/85 (Tables II and III). Four isolates Asia 1 I N D 8/79, Asia 1 R A B 6 4 / 8 5 , Asia 1 I N D 11/80 and Asia 1 A P N 6/87 were found to be divergent with anti-140S guinea pig serum, while Asia 1 63/72, Asia
1 A P N 6/87 and Asia 1 I N D 50/80 were significantly divergent (p < 0.01) with bovine
serum. T h u s , 23 strains out of 26 studied were observed to be nondivergent (not
significantly different: r' < 1 at p = 0.01) from Asia 1 W B N 117/85 by virtue of
serum neutralisation ratios obtained with serum from vaccinated cattle.
As for the other two strains employed for reference, the Tamil N a d u strain (Asia
1 I N D 11/80) presented a broader spectrum t h a n the Rajasthan strain (Asia 1 R A B
64/85). While 13 strains out of 26 studied showed divergence from Rajasthan strain,
18 strains were heterologous to the Tamil N a d u strain. Gujarat isolates appeared to
be closer to Asia 1 I N D 11/80 (Southern region), t h a n to the strain Asia 1 RAB 64/85
which is from the Northwestern region of the country. However, strains from the
state of Rajasthan gave 'r' values > 1, exhibiting homology with Asia 1 R A B 64/85
which originated in the same state.
Evidence of antigenic variation a m o n g Indian type Asia 1 viruses based o n
complement fixation test results employing guinea pig hyperimmune serum against
live F M D virus (5) has been presented in detail by Rai (6) and Rai and Goel (7).
However, the preference for the serum neutralisation test using serum against
inactivated and purified 140S antigen for strain differentiation is well documented
(8) and endorsed by the P e r m a n e nt Subcommittee on F M D of the International
Association of Biological Standardisation (2).
Results obtained in our laboratory show that cross neutralisation of field strains
with serum from vaccinated cattle simulates the performance of candidate or vaccine
strains, as expected from incorporation in the vaccine.
Strain Asia 1 W B N 117/85 had the broadest antigenic spectrum. Except for the
strain from N i z a m a b ad (Andhra Pradesh, 1987), all appear to be closely related to
this candidate vaccine strain.
These studies show that there is considerable antigenic variation among Indian
type Asia 1 strains, with some evidence of antigenic drift over the years. However,
777
uncontrolled livestock movements over vast territories might be responsible for
antigenic divergence a m o n g strains which are contemporary in time and space. The
strain Asia 1 W B N 117/85 exhibited a b r o a d antigenic spectrum and has been
recommended for incorporation in the quadrivalent vaccine.
The antigenic relationship of field strains, originating from a particular region,
to vaccine strain varies from homologou s to divergent in n a t u r e . This observation
indicates the necessity of continuous monitoring of field strains and incorporating
more t h a n one virus strain of a particular virus type in vaccine to control the disease,
depending on the serological spectrum of candidate virus strains.
ACKNOWLEDGMENTS
This work was carried out under the project " C a t a l o g u i n g and strain
differentiation of F M D v i r u s " . Acknowledgments are due to the National Dairy
Development Board, A n a n d (Gujarat), India, for providing financial assistance. The
authors also wish t o t h a n k the F o o t and M o u t h Disease Virus Typing Centre of All
India Coordinated Research Project at H a r y a n a Agricultural University at Hissar,
and the Indian Veterinary Research Institute, Bangalore, for providing the virus
strains.
* *
DIFFÉRENCIATION ENTRE LES SOUCHES DE TYPE ASIA 1 DU VIRUS APHTEUX
ISOLÉES EN INDE. - L.M. Belwal, V.A. Srinivasan et Rama Kant.
Résumé : Les variations antigéniques entre les souches de type Asia 1 du virus
aphteux isolées en Inde ont été étudiées à l'aide d'une microméthode de
neutralisation bidimensionnelle. Deux souches vaccinales et trois souches
sauvages ont été utilisées comme souches de référence. Des variations
antigéniques considérables ont été constatées entre les souches. Tandis que la
souche vaccinale Asia 1 IND 8/79 présentait un spectre antigénique étroit, la
plupart des souches ont pu être apparentées à Asia 1 63/72. Un spectre étendu
a été observé pour la souche Asia 1 WBN 117/85, dont l'incorporation dans
le vaccin quadrivalent a été depuis lors recommandée.
MOTS-CLÉS : Aphthovirus - Fièvre aphteuse - Inde - Variations antigéniques.
*
* *
DIFERENCIACIÓN ENTRE LAS CEPAS DE TIPO ASIA 1 DEL VIRUS AFTOSO
AISLADAS EN INDIA. - L.M. Belwal, V.A. Srinivasan y Rama Kant.
Resumen: Las variaciones antigénicas entre las cepas del tipo Asia 1 del virus
aftoso aisladas en India se han estudiado mediante una microtécnica de
neutralización bidimensional. Se utilizaron dos cepas vacunales y tres cepas
salvajes como cepas de referencia, comprobándose considerables variaciones
antigénicas entre ellas. En tanto que la cepa vacunal Asia 1IND 8/79 presentaba
un espectro antigénico estrecho, la mayoría de las cepas pudieron aparentarse
778
con Asia 1 63/72. En la cepa Asia 1 WBN 117/85, se observó un espectro amplio,
recomendándose su incorporación en la vacuna cuadrivalente.
PALABRAS CLAVE: Aftovirus - Fiebre aftosa - India - Variaciones
antigénicas.
*
* *
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