The document summarizes a study that analyzed genetic variation in isolates from a 2013-2014 foot-and-mouth disease outbreak in South Africa using next-generation sequencing. Key findings include: 1) The most variable genomic regions coded for immune response repression and antigenicity, while the most conserved regions involved capsid stability and replication efficiency. 2) Phylogenetic analysis showed that outbreak isolates clustered within a single genotype of topotype I and were grouped by collection location, indicating two possible transmission pathways. 3) Twenty-two amino acid sites underwent negative selection, while one underwent positive selection and should be further examined for vaccine impact. The study identified genetic variation during the outbreak and implications for regularly updating SAT2

1. BACKGROUND
 Foot-and-mouth disease (FMD):
 Highly contagious viral disease
 Cloven-hoofed animals (e.g. cattle & goats)
 Economic losses: “stamping out” & trade restrictions
 FMD viral genome: open reading frame (ORF) + two
untranslated regions (UTRs)
 ORF: structural proteins (SPs) + non-structural proteins (NSPs)
 Benefits of next-generation sequencing (NGS):
 Insight into emergence of new strains & serotypes
 Epidemiological tracing of FMD viral transmission
2013/14 OUTBREAK
 Location: Bushbuckridge, Mpumalanga Province, RSA
 First clinical signs: 6 August 2013
 Probang and vesicular epithelial samples
 Officially resolved: 10 February 2015
STUDY & AIMS
 Isolates (n=10) from 2013/14 SAT2 FMD outbreak underwent
NGS to generate ORF sequences
 Aims:
 Characterize genetic variation in genome over course
of outbreak
 Determine genome regions that underwent
greatest change
 Determine phylogenetic relationships
 Determine whether selection occurred
RESULTS & DISCUSSION:
VARIATION IN REGIONS
 Most variable regions (non-synonymous):
 Leader (1.17% relative to region length)
o Leader protein: represses innate host immune
response to FMDV  virus can replicate in cell
without obstruction
 Viral protein 1 (VP1) (0.93%)
o VP1: most antigenic SP exposed to exterior of virus particle
 could affect vaccine efficacy
 Least variable regions (non-synonymous):
 VP4 (SP), 2A (NSP), 3B (NSP) & 3C (NSP) (all: 0% change)
o VP4: only SP located internally & not exposed to
outside of virus capsid  no effect on antigenicity
o VP4, 2A & 3C: capsid stability & assembly
o 2A, 3B & 3C: optimal viral replication
o 3C: represses innate host immune
response
Figure 1: Ruptured vesicles in the mouth of cattle infected
with foot-and-mouth disease virus (FMDV)
RESULTS & DISCUSSION:
PHYLOGENETIC ANALYSIS
SAT2/SAR/13/13
SAT2/SAR/10/13
SAT2/SAR/9/13
SAT2/SAR/8/13
SAT2/SAR/4/13
SAT2/SAR/1/13
SAT2/SAR/5/13
SAT2/SAR/6/13
SAT2/SAR/4/14
SAT2/SAR/15/13
SAT2/KNP/19/89
SAT2/SRHO/1/65
SAT2-2 106/67 iso25
Topotype I
Topotype ISAT2/ZIM/22/2003
SAT2/ZIM 004/2002
SAT2/BOT-BUFF/2/69
SAT2/BOT-BUFF/2/68
SAT2/ZAM/18/2009
Topotype III
SAT2/BOT-BUFF/107/72
SAT2/BOT-BUFF/17/69
Topotype II
Topotype IISAT2/BOT-BUFF/170/74
Topotype IISAT2/ZIM/7/83 clone
SAT2/ZIM/11/91
SAT2/NR/1/64
SAT2-1rhod/48 iso26
Topotype III
Topotype IVSAT2/ETH/1/90
Topotype IVSAT2/TAN/5/2012
Topotype IVSAT2/KEN/K137/2014
SAT2/KEN 002/2002
Topotype XSAT2/UGA/1/07 Buffalo 10 QE
SAT2/UGA 002/2002
Topotype XSAT2/UGA-BUFF/12/70
Topotype XSAT2/UGA-BUFF/24/70
Topotype XSAT2/UGA/2/07 Buffalo 6 QE
Topotype IXSAT2/KEN/3/57
Topotype XSAT2/3kenya/21
SAT2/GHA/Tul/5/2018
SAT2/GHA/Tul/2/2018
SAT2/NIG/1/14
Topotype V
SAT2/EGY/24/2014
SAT2/PAT/1/2012
SAT2/EGY/3/2012
SAT2/EGY/9/2012
Topotype VIII
100
70
100
100
100
70
100
100
96
96
100
100
100
100
100
94
100
99
71
100
100
100
94
88
100
89
82
100
88
68
100
81
98
Figure 4: Maximum- likelihood Tree
depicting ORF sequences of the SAT2
FMD strains isolated from cattle in the
FMD Protection Zone. Scale bar
indicates 0.05 substitutions/site. All outbreak
viruses cluster within a single genotype within
topotype I.
Key:
Collection location A
Collection location B
Collection location C
Collection location D
Collection location E
 2013/14 outbreak isolates:
 Cluster within Topotype I
 Grouped together according to collection location
o 2 possibly separate transmission pathways:
Location A  B & C, & Location A  D & E
o May be direct/indirect transmission at
dip tank or within same herd
CONCLUSIONS
 Most variable regions code for
repressing innate cellular immunity
& affect viral antigenicity
 Most conserved regions involved in capsid
stability and assembly, viral replication
efficiency & repressing innate host cellular
immune responses
 Phylogenetic analysis showed outbreak
isolates cluster together into topotype I &
grouped together according to collection
location
 Amino acid substitution & selection
found 23 variable amino acid sites,
with one that underwent positive
selection
IMPLICATIONS
 SAT2 FMDV undergoes genetic
variation during an outbreak
 Identified two highly variable SAT2
genome regions & variable amino acid
substitution sites
 Shows importance of regularly updating and
changing SAT2 FMDV vaccines to include viruses
with suitable antigenic properties
 Shows that disease surveillance, having
capacity to perform diagnostic testing and
characterization of more than one
outbreak virus during single outbreak
are important for present and future
outbreak analyses.
Figure 2: Ruptured vesicles on
the hooves of cattle infected
with foot-and-mouth disease
virus (FMDV)
SEQUENCE AND PHYLOGENETIC ANALYSIS OF THE 2013/14 SAT2
FMD OUTBREAK IN MPUMALANGA,SOUTH AFRICA
Blight, D.1,2, van Heerden, J.2, Heath, L.2, Blignaut, B.1, Fosgate, G.T.1
1Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, 0110, South Africa;
2Transboundary Animal Diseases, Agricultural Research Council – Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa
Figure 3: Schematic diagram of the foot-and-mouth disease (FMD) viral genome.
5’ UTR 3’ UTR
ORF
Leader
VP4
VP3
VP2
VP1
2A
2B
2C
3A
3B
3C
3D
Structural proteins Non-structural proteins
RESULTS & DISCUSSION:
AMINO ACID SUBSTITUTIONS
& SELECTION
Table 1: Summary of amino acid substitutions in foot-and-mouth
disease (FMD) SAT2 2013/14 outbreak isolates
Outbreak
Isolate
Codon start given as amino acid position in respective
region
Leader
VP
2
VP3 VP1
6 75 107 129 152 177 193 124 131 136 26 50
Day 0
Consensus
C N D T N A R A T K K N
SAR/1/13 . . . . . . . . . . . .
SAR/4/13 . . . . . . . . . . . .
SAR/5/13 . . . . . . . . . . . .
SAR/6/13 . . . . . . . . . . . .
SAR/8/13 . . . . . . . . . R . .
SAR/9/13 . . . . . . . . . R . S
SAR/10/13 . . . . . . . . . R . S
SAR13/13 R . . . . . . . . R . S
SAR/15/13 . D G A . . C . A . R .
SAR/4/14 . . G . S V . T . . R S
Table 1 (continued)
Outbreak
Isolate
Codon start given as amino acid position in respective
region
VP1 2B 2C 3A 3D
148 155 175 201 128 75 83 128 46 87 278
Day 0
Consensus
A S E N I G V K N V I
SAR/1/13 . . . . . . I . . . .
SAR/4/13 . F . . . E . . . . .
SAR/5/13 . . . . . . . . . . .
SAR/6/13 . . . . . . . . . . .
SAR/8/13 . . . . . . . . . A .
SAR/9/13 . . . . . . . . . A T
SAR/10/13 . . K . . . . . . A .
SAR13/13 . . K . . E . Q . A .
SAR/15/13 . . . S . E . . D . .
SAR/4/14 T . . S V E . . D . .
The shaded block shows an amino acid position that underwent positive
diversifying selection; all other positions showed negative, purifying
selection
 22 amino acid sites underwent negative selection
o Purifying selection
 1 amino acid site underwent positive selection
o Diversifying selection
 These amino acid substitution sites should be
investigated further to determine if they have
significant impact on vaccine efficacy
METHODS
 Sample preparation for NGS:
 Isolates passaged in cells (PK or IB-RS-2)
 RNA extraction  RT-PCR of ORF  NGS
 NGS data analysis:
 ORF consensus sequence for each isolate
 Aligned isolates to Day 0 consensus sequence & compared
 Identified synonymous & non-synonymous substitutions
 Phylogenetic analysis