FOOT AND MOUTH DISEASE VIRUS EXPRESSING CHIMERIC CAPSID
PROTEIN: A TOOL FOR DELINEATION OF NEW ANTIGENIC SITES AND VACCINE
STRAIN SELECTION
Jitendra Kumar BiswalScientist
ICAR‐Directorate of FMD, Mukteswar, India
Overview
• Global distribution of FMDV serotype A andrecent spread of Genotype VII (18)
• Lack of suitable vaccine strain against currentgenotype VII (18)
• Chimeric FMDV and evidences that the VP2capsid protein has been responsible for theantigenic un‐relatedness of the recent genotype‐18
• Identification new putative antigenic epitope(VP2‐74) and its role for antigenic un‐relatedness.
Global Distribution of Serotype A
Genotype VII (18)As, E (1984‐2010)Indian subcontinent
Genotype IV (10)As, E (1964‐1990)Middle East, South, Central & Northern Asia
Genotype III (4)As (1958‐1964)China
Genotype IX (20)As (1987‐2010)South‐East & East Asia
Genotype VI (16)As (1982‐2001) India
Genotype VIII (19)As (1987‐1991)Middle East
Genotype 25As (2002‐2007)Middle East
Genotype 26(A IRN‐05)As (2001‐2008)Middle East
Genotype 8As (1960)Thailand
Genotype 9Af (1964‐1974)East & North AfricaGenotype 15(Af (1977‐2006)East & West Africa
Genotype 23Af (1997‐1998)North‐West Africa
Genotype 11Af (1965)KenyaGenotype 1
E (1929)Germany
Genotype 5SA (1958‐1967)Northern South America
Genotype I (2)E, SA, As, Af (1932‐1989)
Genotype 12SA (1966‐1971)Argentina
Genotype 13SA (1968‐2002)Southern South America
0.02 nucleotide substitution/site
ASIA Topotype
AFRICA Topotype
EURO‐SOUTH AMERICATopotype
76
100
99
9999
100
100
91
99
100
99
9968
10071
87 1001008496
95
8263
62
Genotype 18 (VII)• Since 2001, genotype VII (18) has been exclusively
responsible for all the field outbreaks
• Within the genotype-18 a divergent and unique lineage emerged in late part of 2002, which showed an amino acid (aa) deletion at 59th position of VP3 (VP359-deletion group). From 2007–2008, there is an upsurge in incidence of outbreaks due to this lineage.
• In 2015, it appeared for the 1st time in the Middle East, during the same year, by Iran, Turkey and Armenia. Most recently (May 2017), this virus was identified in Northern Israel, on the Lebanese and Syrian borders, Nepal and Bhutan.
Antigenic relatedness• Currently, majority of the field isolates belonging
to the VP359-deletion group were foundantigenically unrelated to the in-use vaccinestrain through the 2D-VNT assay.
• Considering the antigenic diversity, a panel of 3candidate vaccine strains were selected, and onestrain provided good antigenic coverage (79 outof 84 tested isolates were matched).
• Sequence analysis in the countrywidelongitudinal data-set could not determine anyspecific fixation of amino acid substitution at theknown antigenically critical positions.
VP4 VP2 VP3 VP1
VP4 VP2 VP3 VP1VP3
VP4 VP2 VP3 VP1VP2
VP4 VP2 VP3 VP1VP4
Chimeric infectious clones were generated through Mega‐primer mediated domain swapping method (Biswal et al., 2015)
VP4 VP2 VP3 VP1 VP4 VP2 VP3 VP1
A IND 40/2000 A PD 26/2015
Domain Swapping Mutagenesis and chimericcDNA clones
VP4 VP2 VP3 VP1VP2
1 146 218
VP4 VP3 VP1
VP4 VP3 VP1
Chimeric VP2
Chimeric VP2
A IND 40/2000A PD 26/2015
Chimeric‐VP2 cDNA clones
00.10.20.30.40.50.60.70.80.91
40/2000 40/2000‐Chi‐VP2 (1‐146)
40/2000‐Chi‐VP2 (146‐218)
r1 Value
s
2D‐VNT with A IND 40/2000 BVS
Position and Amino Acid
Secondary Structure
Entropy Values
ConsurfValues
Consensus between genetic and antigenic variants
55-V/K/E/L/N/T αZ helix 0.403 -0.340 Not found74 -A/P/S βC–βC loop 0.798 3.033 Yes (A→P)
79-E/V/G/A/Q βC strand 0.505 2.533 No96 -D/G/N/K/E αA helix 0.510 0.332 Not found
131-E/K/D/G/H/N βE–βF loop 1.064 1.734 Not found
Putative antigenic residue(s) on the VP2‐protein of the antigenic variants
VP2‐72
VP2‐74
VP2‐131
Reduction in Virus neutralization titre and associated ‘r1’‐value after site‐directed
mutagenesis
0
20
40
60
80
100
A 40/2000 A 40/2000-A2074P
A 40/2000-A2074S
A 40/2000-K2131E
Red
uct
ion
in V
N ti
tre
(%)
2D-VNT was carried out using BVS against A IND 40/2000
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
A 40/2000 A 40/2000-A2074P
A 40/2000-A2074S
A 40/2000-K2131E
r1-V
alue
s
A B
r1‐values of serotype‐A field isolates with Rabbit anti‐146S serum
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
A 40/2000-Chi-VP2 A 40/2000
r1-v
alue
sof
fiel
d is
olat
es
18/20 1/20
Conclusions
• Reverse genetics technology based chimericFMDV is a handy tool for the determination ofthe role of individual capsid protein(s) in theviral‐antigenicity .
• New antigenic epitope (VP2‐74) was identifiedon the capsid surface of FMDV serotype A‐genotype VII(18).