1.
GENETIC AND ANTIGENIC
VARIATION OF FOOT-AND-MOUTH
DISEASE VIRUS DURING
PERSISTENCE IN NATURALLY
INFECTED CATTLE AND BUFFALO
Jitendra Kumar Biswal
Scientist
ICAR-Directorate of Foot-and-mouth disease, Mukteswar,
India

2.
Need to study FMDV persistence in
endemic settings
• Better understanding of FMDV natural ecology
for development of effective control
strategies.
• Possible roles of persistently infected animals
in initiating new outbreaks
• Quantification of risk posed by carriers
• Enhance our understanding on the basic
biology of FMDV

3.
ABIS Dairy, Rajnandagaon, Chhattisgarh
Biggest Dairy in Central India; Total Population= 5000; animals on milch = 1836

4.
FMD outbreak summary at ABIS Dairy
• Routine Vaccination
• Index case on 24th
December 2013.
• 222 clinical cases out
of 1836 animals over
a course of 35 days of
infection, with an
cumulative incidence
rate : 12.1%

5.
Study Design
• Up to total 21 clinically affected and 16
asymptomatic cattle ; 11 clinically affected and
6 asymptomatic buffalo were sampled for O-P
fluids at 2-3 months interval for a period of 13
months.
• Viruses were rescued from OPF in LFBK-αVβ6
cells and the entire capsid coding (P1)
sequences were determined from the cell
culture supernatant at low passage level (3-4).

6.
Genome Detection
0%
20%
40%
60%
80%
100%
120%
3Month 5month 7month 10month 13month
Clinical Cattle Asymptomatic cattle
%positive
Study Period
0
20
40
60
80
100
120
3 Month 5 month 7 month 10 month 13 month
Clinical Buffalo Asymptomatic Buffalo
%positive
Study Period

7.
Virus Isolation
0%
10%
20%
30%
40%
50%
60%
70%
80%
3 Month 5 month 7 month 10 month 13 month
Clinical Cattle Asymptomatic cattle
Study Period
%positive
0
10
20
30
40
50
60
70
80
90
3 Month 5 month 7 month 10 month 13 month
Clinical Buffalo Asymptomatic Buffalo
Study Period
%positive

8.
Virus Isolation on LFBK-αVβ6 cell line
SL No Species Status ID Mar-14 May-14 Aug-14 Nov-14 Feb-14
1 CATTLE Asymptomatic ABIS 3595 P4 P3 Neg Neg Neg
2 CATTLE Asymptomatic ABIS 3512 P4 Neg Neg Neg Neg
3 CATTLE Asymptomatic ABIS 3568 P4 P3 Neg Neg Neg
4 CATTLE Asymptomatic ABIS 3035 P4 Neg Neg Neg Neg
5 CATTLE Clinical ABIS 458 P4 Neg Neg Neg Neg
6 CATTLE Clinical ABIS 5430 P4 Neg Neg Neg Neg
7 CATTLE Clinical ABIS 6636 P4 Neg Neg Neg Neg
8 CATTLE Clinical ABIS 12517 P4 P3 Neg Neg Neg
9 CATTLE Clinical ABIS 6646 P4 Neg Neg Neg Neg
10 CATTLE Clinical ABIS 5143 P4 Neg Neg Neg Neg
11 CATTLE Clinical ABIS 6666 P4 Neg Neg Neg Neg
12 CATTLE Clinical ABIS 5173 P4 Neg Neg Neg Neg
13 CATTLE Clinical ABIS 20252 P4 Neg Neg Neg Neg
14 CATTLE Clinical ABIS 20213 P4 Neg P3 Neg Neg
15 CATTLE Clinical ABIS 20453 P4 Neg Neg Neg Neg
16 CATTLE Clinical ABIS 20216 P4 Neg Neg Neg Neg
17 BUFFALO Asymptomatic ABIS B2026 Neg P3 Neg Neg
18 BUFFALO Asymptomatic ABIS B1932 Neg Neg P4 Neg
19 BUFFALO Asymptomatic ABIS B1765 Neg P3 Neg Neg
20 BUFFALO Asymptomatic ABIS 1961 P3 Neg Neg Neg
21 BUFFALO Asymptomatic ABIS B2017 P3 Neg Neg Neg
22 BUFFALO Clinical ABIS 1760 P4 Neg Neg Neg Neg
23 BUFFALO Clinical ABIS 16403 P4 Neg Neg P3 P3
24 BUFFALO Clinical ABIS 1964 P4 P3 P3 P4 Neg
25 BUFFALO Clinical ABIS 2224 P4 Neg Neg P4 Neg
26 BUFFALO Clinical ABIS 1908 P4 Neg Neg Neg Neg
27 BUFFALO Clinical ABIS 2036 P4 Neg Neg Neg Neg
28 BUFFALO Clinical ABIS 1854 P4 P3 P3 Neg Neg
29 BUFFALO Clinical ABIS 2067 P3 Neg Neg Neg
30 BUFFALO Clinical ABIS B1937 Neg P3 Neg Neg
31 BUFFALO Clinical ABIS B1816 P3 P3 Neg Neg

9.
ML tree depicting
the phylogentic
relationships of
carrier and acute
viruses of abis
farm
ABIS/1765/AUG/2014/Carrier
ABIS/1937/AUG/2014/Carrier
ABIS/1932/AUG/2014/Carrier
ABIS/2067/MAY/2014/Carrier
ABIS/1816/AUG/2014/Carrier
ABIS/1964/NOV/2014/Carrier
ABIS/16403/FEB/2015/Carrier
ABIS/1908/MAR/2014/Carrier
ABIS/2017/MAY/2014/Carrier
ABIS/2036/MAR/2014/Carrier
ABIS/1854/MAR/2014/Carrier
ABIS/1854/MAY/2014/Carrier
ABIS/1854/AUG/2014/Carrier
ABIS/1964/MAR/2014/Carrier
ABIS/1964/MAY/2014/Carrier
ABIS/2224/MAY/2014/Carrier
ABIS/1961/MAY/2014/Carrier
ABIS/1760/MAR/2014/Carrier
ABIS/2224/MAR/2014/Carrier
ABIS/6666/MAR/2014/Carrier
ABIS/A421/JAN/2014/Acute
ABIS/6670/JAN/2014/Acute
ABIS/A5636/JAN/2014/Acute
ABIS/6684/JAN//2014/Acute
ABIS/5430/MAR/2014/Carrier
ABIS/16403/MAR/2014/Carrier
ABIS/458/MAR/2014/Carrier
ABIS/20213/MAR/2014/Carrier
ABIS/5173/MAR/2014/Carrier
ABIS/6636/MAR/2014/Carrier
ABIS/12517/MAR/2014/Carrier
ABIS/12517/MAY/2014/Carrier
ABIS/3512/MAR/2014/Carrier
ABIS/20252/MAR/2014/Carrier
ABIS/3035/MAR/2014/Carrier
ABIS/3595/MAY/2014/Carrier
ABIS/3595/MAR/2014/Carrier
ABIS/20453/MAR/2014/Carrier
ABIS/3568/MAR/2014/Carrier
ABIS/3568/MAY/2014/Carrier
ABIS/20213/AUG/2014/Carrier
IND339/2013
BHU/1/2013
LIB/2/2013
SAU/3/2013
Ind2001d
IND120/2002
IND74/2002
Ind2001b
Ind2001
IND331/2007
IND320/2007
IND408/2007
Pan Asia
Vaccine strainINDR2/1975
98
100
100
100
100
100
100
100
100
100
73
79
0.02

10.
Nucleotide
divergence of virus
from carrier
animals compared
to virus collected
during acute phase
of infection
Mean dN/dS (ω)= 0.188
Animal
No
Mar’14 May’14 Aug’14 Nov’14 Feb’15 dN/dS
Convalescent Cattle
458 0.02 - - - -
5430 0.03 - - - -
6636 0.06 - - - -
6666 0.04 - - - -
12517 0.05 1.3 - - - 0.183
20213 0.09 - 0.08 - -
20453 0.06 - - - -
20252 0.07 - - - -
5173 0.07 - - - -
Asymptomatic Cattle
3035 0.07 - - - -
3512 0.06 - - - -
3568 0.08 1.00 - - - 0.357
3595 0.05 0.07 - - - 0.069
Convalescent Buffalo
1760 0.04 - - - -
1816 - - 1.0 - -
1854 0.01 0.07 0.05 - - 0.194
1908 0.03 - - - -
16403 0.02 - - - 1.0 0.159
1937 - - 0.08 - -
1964 0.04 0.06 - 1.2 - 0.142
2036 0.02 - - - -
2067 - 0.05 - - -
2224 0.02 1.00 - - - 0.076
Asymptomatic Buffalo
1765 - - 1.0 - -
1932 - - 1.1 - -
1961 - 0.06 - - -
2017 - 0.07 - - -
2.4%

11.
Distribution of synonymous/non-synonymous variations at P1 region
VP4 (1-85) VP2(86-303) VP3 (304-523) VP1(525-734)

12.
Genetic Variation at Herd Level
• Overall, in the capsid coding region, a total 304 sites
(13.8%) were found to be polymorphic, of which 208
sites showed single nt polymorphism (9.4%) and 96
sites were shared by more than 2 nts (4.3%).
• Among the base substitutions, maximum (86%) were
transitions and 14% were transversions.
• The capsid protein in carrier viruses showed
variations at 75 aa positions (10.2%) of which 66
positions (8.9%) were occupied alternately by two aa
and 9 positions (1.2%) were substituted by more
than two aa.
• 30 sites: VP1, 23 sites: VP3 and 17 sites: VP2.

13.
Selection Pressure Analysis
• Two codons in VP1 (138 and 148) and one codon each in
VP2 (78) and VP3 (76) to be under positive selection with
statistical significance as determined by SLAC, FEL and IFEL
methods.
• Additionally, codon 73 in VP3 was found to be under
selection pressure by SLAC and FEL.
• A total of 28 codons was found to experience episodic
diversifying selection. Of which, 10 codons were in VP3
followed by 9 in VP1 and 8 in VP2.
• VP4 revealed one codon (61) to be under episodic selection

14.
Statistical parsimony analysis of capsid coding sequences of
acute and carrier viruses
May’14 : CC12517 and CB2224, Aug’14 : CB1816 and AB1932, Nov’14: CB1964 and
Feb’15: CB16403 have >1% nucleotide divergence from acute virus

15.
Evolutionary rates
• By using the relaxed lognormal molecular clock model, the
mean nucleotide substitution was estimated to be was
estimated to be 1.816 x 10-2 substitution/site/year (s/s/y)
with a 95% CI: 1.362-2.31 x 10-2 s/s/y.
• The average mutation rate of codon positions 1+2 and 3
was estimated to be 0.519 and 1.963, respectively.
• This substitution rate is significantly faster than the rates
reported earlier for Ind2001 lineage of serotype O virus
Indian virus isolates over a period of 12 years (6.338 x 10-3
s/s/y).
• This clearly indicates higher selection pressure on virus
genome during persistent infection

16.
Antigenic relationship (r-value) obtained in
2D-MNT of the acute and carrier viruses
Jan’14 March’14 May’14 Aug’14 Nov’14 Feb’15
C6670 (acute) 0.6
C5636 (acute) 0.6
Carrier virus
CC12517 0.33 0.522
CC5143 0.78
CC20252 0.32
CC20213 0.36 0.41
CB16403 0.66 0.21
CB1964 0.41 0.383 0.396 0.173
CB2224 0.33 0.22
CB1908 0.33
CB1854 0.33 0.143 0.277
AC3595 0.49 0.295
AC3512 0.33
AC3568 0.23 0.819
AC3035 0.66
AB1932 0.229
AB2017 0.248

17.
Location of Putative antigenic sites on
neutralization-resistant FMDV
VP1-13 (T-S), VP1-54 (M-L), VP1-143 (N-K), VP3-123 (I-V), VP3-131 (E-A), VP3-203 (A-V)

18.
Location of amino acids possibly responsible for high antigenicity
2194 A-P
3166 D-G

19.
Summary
• Mutations at the rate of 1.816 x 10-2 substitution/site/year
(s/s/y).
• Fixation of nucleotide and amino acid changes were observed
at some position, majority were not conserved in consecutive
isolates.
• Between different animals, mean dN/dS (ω) value of the
entire capsid coding region varies from 0.076 to 0.357.
• From the statistical parsimony analysis, it was evident that all
the virus isolates from carrier animals were originated from acute
virus except six isolates.
• The antigenic relationship value indicates fluctuation of
antigenic variants in carrier animals.
• Evidence of viral activity in the persistently infected animals
under field scenario and probable role of host factor in shaping their
evolution.

20.
• ABIS Dairy, Raipur, India.
•ICAR
• USDA
•All the Scientific and technical staffs
of ICAR-Directorate of FMD
•
ACKNOWLEDGEMENTS