Journal of Applied Animal Research

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/taar20

Detection and identification of foot-and-mouth

disease O/ME-SA/Ind-2001 virus lineage, Indonesia,
2022

Edy Budi Susila, Rosmalina Sari Dewi Daulay, Dewi Noor Hidayati, Sapto Rini
Budi Prasetyowati, Wriningati, Ernes Andesfha, Sri Handayani Irianingsih, I.
Nyoman Dibia, Faisal, Arif Supriyadi, Yuni Yupiana, Muhammad Muharram
Hidayat, Nuryani Zainuddin & Hendra Wibawa

To cite this article: Edy Budi Susila, Rosmalina Sari Dewi Daulay, Dewi Noor Hidayati, Sapto
Rini Budi Prasetyowati, Wriningati, Ernes Andesfha, Sri Handayani Irianingsih, I. Nyoman Dibia,
Faisal, Arif Supriyadi, Yuni Yupiana, Muhammad Muharram Hidayat, Nuryani Zainuddin &
Hendra Wibawa (2023) Detection and identification of foot-and-mouth disease O/ME-SA/
Ind-2001 virus lineage, Indonesia, 2022, Journal of Applied Animal Research, 51:1, 487-494,
DOI: 10.1080/09712119.2023.2229414

To link to this article: https://doi.org/10.1080/09712119.2023.2229414

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and Animal Health Services. Published by
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Francis Group

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JOURNAL OF APPLIED ANIMAL RESEARCH
2023, VOL. 51, NO. 1, 487–494
https://doi.org/10.1080/09712119.2023.2229414

Detection and identification of foot-and-mouth disease O/ME-SA/Ind-2001 virus

lineage, Indonesia, 2022
Edy Budi Susila a, Rosmalina Sari Dewi Daulaya, Dewi Noor Hidayati a, Sapto Rini Budi Prasetyowatia, Wriningatia,
Ernes Andesfhab, Sri Handayani Irianingsih c, I. Nyoman Dibiad, Faisale, Arif Supriyadif, Yuni Yupiana g,
Muhammad Muharram Hidayatg, Nuryani Zainudding and Hendra Wibawa c
a
Pusat Veteriner Farma/National Center for Veterinary Biologics, Surabaya, Indonesia; bNational Veterinary Drug Assay Laboratory, Balai Besar
Pengujian Mutu dan Sertifikasi Obat Hewan, Bogor, Indonesia; cBalai Besar Veteriner Wates/Disease Investigation Center Wates, Yogyakarta,
Indonesia; dBalai Besar Veteriner Denpasar/Disease Investigation Center Denpasar, Denpasar, Indonesia; eBalai Veteriner Medan/Disease
Investigation Center Medan, Medan, Indonesia; fBalai Veteriner Banjarbaru/Disease Investigation Center Banjarbaru, Banjarbaru, Indonesia;
g
Directorate Animal Health, Directorate General of Livestock and Animal Health Services, Jakarta, Indonesia

ABSTRACT ARTICLE HISTORY

Foot-and-mouth disease (FMD) is a contagious disease affecting cloven-hoofed animals, which has been Received 6 February 2023
well-documented as one of the major animal diseases that causes severe economic loss in livestock Accepted 21 June 2023
sectors. The disease is endemic in many countries, particularly in Asia and Africa. Indonesia has been
KEYWORDS
declared a disease-free country since 1986, and the World Organization of Animal Health (WOAH/OIE) Foot-and-mouth disease;
recognized Indonesia as an FMD-free country without vaccination in 1990. However, the FMD virus serotype; O/ME-SA/Ind-2001;
was detected in many disease outbreaks in cattle and goats in Indonesia in May 2022. This study phylogenetic analysis;
reports the detection and identification of FMD serotype O viruses in Indonesia. Although these Indonesia
viruses appeared to belong to the ME-SA/Ind-2001e lineage, they formed a unique cluster with 95.3%
average nucleotide sequence similarity of the FMD VP1 gene to Ind-2001e viruses from other Asia
countries. The illegal trade of live animals from endemic areas in Southeast Asia is one of the possible
routes regarding the incursion of FMD in Indonesia, however, it requires further investigation.

Introduction phylogenetic relationships and a nucleotide difference of

Foot-and-mouth disease (FMD) is a contagious animal disease <5% based on VP1 protein (Hemadri et al. 2002; Knowles and
caused by an RNA virus from Picornaviridae, genus Aphthovirus, Samuel 2003). The O/ME-SA/PanAsia lineage emerged from
species foot-and-mouth disease virus. The virus has seven ser- the Indian continent (Bangladesh, Bhutan, India and Nepal). It
otypes, including O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3, and is became the major serotype-O virus lineage that spread globally
further divided genetically into topotypes and lineages into the Middle East, Southeast Asia, North and South Africa,
(Grubman and Baxt 2004). The disease affects cloven-hoofed and Europe between 1990 and 2000 (Hemadri et al. 2002;
animals causing a severe economic loss in livestock sectors Knowles et al. 2005; Bachanek-Bankowska et al. 2018). The
and is endemic in many countries, particularly in Asia and Ind-2001 lineage of O/ME-SA topotype (O/ME-SA/Ind-2001),
Africa (Brito et al. 2015). To date, there are 10 topotypes of ser- again, was initially identified in the Indian subcontinent in
otype O identified, including Europe-South America (Euro-SA), 2001 as a distinct group from the predominant PanAsia
Middle East South Asia (ME-SA), South East Asia (SEA), Cathay lineage (Hemadri et al. 2002; Knowles et al. 2005; Bachanek-
(CHY), West Africa (WA), East Africa 1 (EA-1), East Africa 2 Bankowska et al. 2018). Since then, the Ind-2001 lineage has
(EA2), East Africa 3 (EA-3), Indonesia-1 (ISA-1) and Indonesia-2 diverged into five sub-lineages (Ind-2001a to Ind-2001e) and
(ISA-2) (Brito et al. 2015; Blacksell et al. 2019). become significant in the Middle East, Asia and Africa (Bacha-
In Southeast Asian (SEA) countries, serotype O has been nek-Bankowska et al. 2018; Blacksell et al. 2019).
recognized as the predominant serotype to cause more FMD Within the Ind-2001 lineage, a large proportion of FMD out-
outbreaks than serotype A and Asia 1 (Brito et al. 2015; Blacksell breaks were due to infection of the Ind-2001d lineage in South
et al. 2019). Within the serotype O, South East Asia (SEA) and Asia, North Africa and Southeast Asia for 2–3 years from 2012 to
Middle East-South Asia (ME-SA) topotypes were frequently 2015. However, viruses belonging to the Ind-2001e lineage
detected in FMD outbreaks in SEA countries (Blacksell et al. have become predominant since 2016 (Bachanek-Bankowska
2019). In particular ME-SA topotype, several virus lineages et al. 2018). Moreover, since 2020 there has been an increasing
have been identified as strains that temporarily close related dominance of O/ME-SA/Ind-2001e lineage in SEA countries,
or belong to unique outbreaks occurring in certain regions including Cambodia, Laos, Myanmar, Vietnam and Thailand
including PanAsia, Ind2001 and Iran2001 based on (King 2021).

CONTACT Dewi Noor Hidayati [email protected] Pusvetma, Jl. A. Yani No 68-70 Surabaya, East Java 60231, Indonesia
© 2023 Directorate General of Livestock and Animal Health Services. Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow
the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
488 E. B. SUSILA ET AL.

FMD is endemic in most Southeast Asia countries except of May 2022 and named the group of the isolates as ISA-22.
Philippines, Singapore and Brunei. In Indonesia, the first FMD Since this was the first case in Indonesia after 35 years free
case occurred in East Java in 1887. Nevertheless, after imple- from FMD, we aimed to identify the genetic lineage of the
menting strict animal movement, strong quarantine measures viruses that cause the FMD outbreaks. This study also intended
and mass vaccination programs, no FMD has reported since to determine the source of the virus introduction and to infer
December 1983, and Indonesia declared a disease-free the molecular pattern of virus transmission amongst areas in
country in 1986 (Blacksell et al. 2019). However, after 35 years Indonesia for a better understanding of disease control.
free from FMD, at the beginning of May 2022, several disease
outbreaks with FMD-like symptoms were reported in Aceh
and East Java provinces, followed by several FMD cases in Material and methods
other areas in Java, Sumatera, Kalimantan and West Nusa Teng-
Clinical samples and diagnostics
gara. This study reports the detection and identification of
FMD-serotype O virus that belongs to the ME-SA/Ind-2001e During the initial outbreaks of disease in Java, Sumatera, Kali-
lineage in Indonesia. In this study, we collected clinical mantan and West Nusa Tenggara in May 2022 (Figure 1), a
samples from FMD outbreaks in Indonesia in the first 2 weeks total of 24 swabs and two blood samples were collected from

Figure 1. Distribution of samples collected from Java, Sumatera, Kalimantan and West Nusa Tenggara for foot and mouth virus characterization. Numbers represent
the sequence of collection dates that are in the same order as ID number in Table 1. Dots represent village-based centroid where samples were collected from FMD
cases in livestock. A zoom-in of FMD case locations in Aceh and North Sumatera provinces and in Java is indicated in the bottom box.
 JOURNAL OF APPLIED ANIMAL RESEARCH 489

cattle and goats (Table 1). These animals showed clinical symp- MA, USA) on the ABI 3130 Genetic Analyser (Applied Biosys-
toms of pyrexia, anorexia and vesicles either in buccal mucous tems, Foster City, CA, USA). DNA sequencing was done using
membranes or between claws and coronary band; and hypersa- clinical samples without prior isolation in cell culture. It was
livation in cattle. The presence of the FMD virus was detected performed using a previously developed protocol (Le et al.
directly from clinical samples (swabs and blood samples) 2012) with modified primers for amplification and sequencing
through routine viral RNA extraction procedures followed by reactions (primer sequences are available upon request).
real-time reverse transcription polymerase chain reaction Genome assembly and analysis were performed using the
(rRT-PCR) technique using primers and probe targeting the AB1 files extension in UGENE v.40 software (https://ugene.
3D gene of FMD virus (WOAH 2021). Using these primers and net/). The VP1 sequence data were submitted to GenBank
probes, we could detect, on average, 92% of FMD cases positive under accession numbers, as shown in Table 1. The
from the total tested animals showing clinical signs (data not maximum likelihood (ML) method with 1000 bootstrap repli-
shown). This study is a collaborative work supported by cates using the fittest and best substitution model of HKY+G
Disease Investigation Centres (DICs) in Medan, Wates, Banjar- (based on the lowest Akaike information criterion/AIK) was
baru, and Denpasar and the Indonesian FMD Reference Labora- used to construct a phylogenetic tree based on 639 nt length
tory, National Centre for Veterinary Biologics (NCVB) in of complete VP1 coding sequence from 26 FMD viruses of
Surabaya and National Veterinary Drug Assay Laboratory this study and 60 FMD virus sequences that available in
(NVDAL), Bogor, Indonesia. GenBank (3 viruses of ISA-1, 2 of ISA-2, and 55 of ME-SA topo-
type viruses). Evolutionary distances were computed using
average pairwise distance within and between sequence
DNA sequencing and phylogenetic analysis groups in MEGA X (Kumar et al. 2018).

This study conducted the molecular genetic assay mainly in the

VP1 region to identify the serotype and lineage of FMD virus.
Results
The Sanger dideoxy sequencing targeting the full length of
VP1 coding region [639 nucleotides (nt) total length] was per- To determine genetic relationships and molecular epidemiol-
formed in the National Centre of Veterinary Biologics (NCVB), ogy of the FMD virus, the viral capsid protein VP1 (encoded
Surabaya and National Veterinary Drug Assay Laboratory by 1D gene) has been widely used for FMD phylogenetic analy-
(NVDAL), Bogor Indonesia, by using the Big Dye Terminator sis (Knowles et al. 2016). Phylogenetic analysis based on the
v3.1 Cycle sequencing kit (Thermo Fisher Scientific, Waltham, VP1 coding sequence showed that all FMD viruses detected

Table 1. Metadata of samples collected from initial FMD outbreaks in Indonesia, May 2022.
Collection Species Sample Ct Accession
ID Strain name date District Province origin origin* value number
1 ISA/Tamiang/01220759-P4/2022 04/05/2022 Aceh Tamiang Aceh Cattle Plasma 26.22 ON783873
2 ISA/Tamiang/01220759-P10/ 04/05/2022 Aceh Tamiang Aceh Cattle Plasma 24.18 ON783874
2022
3 ISA/Lamongan/PVT-VL6/2022 04/05/2022 Lamongan East Java Cattle Oral 17.85 ON854936
4 ISA/Sidoarjo/PVT/2022 04/05/2022 Sidoarjo East Java Cattle Oral 22.84 ON854937
5 ISA/Mojokerto/PVT/2022 04/05/2022 Mojokerto East Java Cattle Oral 17.19 ON854938
6 ISA/Babel/PVT-AN/2022 07/05/2022 Bangka Selatan Bangka Belitung Cattle Oral 19.74 ON854939
7 ISA/Langkat/A01220761/2022 08/05/2022 Langkat North Sumatera Cattle Oral 31.05 ON854934
8 ISA/DeliSerdang/A01220762/ 08/05/2022 Deli Serdang North Sumatera Cattle Oral 24.39 ON854935
2022
9 ISA/Probolinggo/PVT/2022 08/05/2022 Probolinggo East Java Cattle Oral 21.58 ON854940
10 ISA/Malang/PVT/2022 08/05/2022 Malang East Java Cattle Oral 22.14 ON854941
11 ISA/Boyolali/A04222562/2022 08/05/2022 Boyolali Central Java Cattle Oral 14.87 ON854942
12 ISA/Pasuruan/A04222583/ 2022 09/05/2022 Pasuruan East Java Cattle Oral 19.08 ON854943
13 ISA/Pangkal Pinang/ PVT/2022 09/05/2022 Pangkal Pinang Bangka Belitung Cattle Oral 21.09 ON854944
14 ISA/LTE/A06220465/2022 09/05/2022 Lombok Tengah West Nusa Cattle Oral 22.26 ON854945
Tenggara
15 ISA/LTI/A06220466/2022 09/05/2022 Lombok Timur West Nusa Cattle Oral 23.20 ON854946
Tenggara
16 ISA/Mempawah/BBR11/2022 09/05/2022 Mempawah West Kalimantan Goat Oral 26.57 ON854947
17 ISA/KWB/A0522106/2022 10/05/2022 Kotawaringin Center Kalimantan Cattle Oral 19.18 ON854948
Barat
18 ISA/Banjarnegara/ A04222582/ 10/05/2022 Banjarnegara Central Java Cattle Foot 20.79 ON854949
2022
19 ISA/Klaten/A04222596/2022 11/05/2022 Klaten Central Java Cattle Oral 17.09 ON854950
20 ISA/Jombang/PVT-J11/2022 11/05/2022 Jombang East Java Goat Oral 14.29 ON854951
21 ISA/Gresik/PVT-KBMS/2022 12/05/2022 Gresik East Java Cattle Oral 24.52 ON854952
22 ISA/Purbalingga/ A04222582/ 12/05/2022 Purbalingga Central Java Cattle Oral 16.86 ON854953
2022
23 ISA/Pemalang/A04222613/2022 12/05/2022 Pemalang Central Java Cattle Oral 17.05 ON854954
24 ISA/Semarang/ A04222614/2022 13/05/2022 Semarang Central Java Cattle Oral 18.53 ON854955
25 ISA/Magetan/A04222620/2022 13/05/2022 Magetan East Java Cattle Oral 20.22 ON854956
26 ISA/HSU/A0522099-5/2022 15/05/2022 Hulu Sungai Utara South Kalimantan Goat Oral 24.09 ON854957
*Plasma was collected from blood samples. Oral samples contained swabs from vesicles or lesion and saliva, whereas foot sample contained swabs of vesicles or lesion
found in foot or coronary band.
490 E. B. SUSILA ET AL.

during the disease outbreaks in Indonesia in May 2022 (named Discussion

ISA-22) belonged to the O/ME-SA/Ind-2001e sequence
This study aimed to identify the genetic lineage of viruses
(Figure 2). Although all the ISA-22 viruses were clustered
responsible for FMD outbreaks, to infer the virus transmission
under the Ind-2001e lineage, they formed a distinct cluster sep-
across regions in Indonesia in the initial phase of the outbreaks.
arated from those of Ind-2001e from other Asian countries with
In combination with recent epidemiological data of FMD out-
95.3% average nucleotide sequence similarity (Table 2). Hom-
breaks in Asia, this study could be used as a preliminary
ology analysis using the BLAST search tool (https://blast.ncbi.
study to obtain possible consideration of the virus introduction
nlm.nih.gov/Blast.cgi) found that the nearest nucleotide
into Indonesia. FMD viruses of O/ME-SA/Ind-2001e were
sequence identity of VP1-FMD that was publicly available in
detected and identified from disease outbreaks with FMD
GenBank was an FMD virus sample from China, XJ/CHA/2017
symptoms in cattle and goats in Indonesia in May 2022. Our
(Ind-2001e virus lineage, Accession Number MF461724), with
study showed that the nucleotide difference of this FMD virus
VP1 nucleotide similarity ranging from 95.6% to 96.2% to ISA-
was >4.7% to the existing O/ME-SA/Ind-2001e viruses that cir-
22 viruses. Sequence analysis using the between-group
culating in ASIA; hence, additional study is necessary to deter-
average pairwise distance indicated low nucleotide identity of
mine the phylogenetic relationships of this Indonesian virus
the VP1 gene of ISA-22 to that of other virus lineages of O/
lineage.
ME-SA topotype, including the earlier Indonesian FMD viruses
We found that there was no significant difference in both
that are now extinct (ISA-1 and ISA-2). By contrast, high nucleo-
nucleotide and amino acid sequences of VP1 protein
tide identity of VP1 sequence (99.6%) was detected within ISA-
amongst virus samples collected from goats and most of
22 viruses, as indicated by a low replication pairwise distance
those collected from cattle. In addition, no amino acid substi-
within this group (0.004) (Table 2).
tutions were found either at the critical amino acid sites
The most recent common ancestral node of the ISA-22 virus
within the VP1 epitope at position 144 (V), 148 (L), 154 (K)
lineage could not yet be determined using the 26 sequences
and 208 (P), or in the position 145–147 of VP1 protein (retained
used for phylogenetic analysis. At first glance from the phyloge-
RGD sequence motif) that involved in the adsorption of FMD
netic tree, three Ind-2001e viruses from Cambodia isolated in
virus to host cell where their alteration more or less affecting
2019 (CAM-19) seemed to have a potential relationship as
antigenic loci and corresponding monoclonal antibody reactiv-
virus ancestors to the ISA-22 group (Figure 2). However, this
ity (Jinding et al. 2006).
was not supported by strong statistical evidence as the boot-
The course of the virus’s incursion into Indonesia remains
strap value in the common node of these two groups was
unknown. We could not yet determine whether the Ind-
less than 50% although the best substitution model (HKY+G)
2001e virus group from Cambodia collected in 2019 (CAM-19)
was used for generating the tree. The more appropriate con-
is a direct precursor of the Ind-2001e virus group in Indonesia
clusion to define this connection is that they are genetically
(ISA-22) since the statistical bootstrap value supporting the
close related. This topology inference was found to be reversed
node that linking these two groups was low. However, the
with the results of molecular analysis of VP1 gene of CAM-19
tree suggests that CAM-19 strains are closely related to those
which revealed only 94.5% nucleotide sequence identity to
circulated in Indonesia or strains circulated in Cambodia in
ISA-22 (Table 2), however, this may be due to the inclusion of
2019 and in Indonesia in 2022 belonging to the same lineage
calculated ORFs and ORF coding.
(Ind-2001e) and probably share a close common ancestor,
Phylogenetic analysis inferred that ISA-22 viruses from North
but emerged at different outbreak events. A recent report
Sumatera (ISA/Langkat/A01220761/2022 and ISA/Deli Serdang/
from The World Reference Laboratory for Foot-and-Mouth
A01220762/22) have likely emerged preceding ISA-22 viruses
Disease (WRLFMD) showed that some virus sequences from
from Aceh (ISA/Tamiang/01220759-P4/2022 and ISA/
Thailand and Malaysia in 2021 were genetically more closely
Tamiang/01220759-P10/2022). Furthermore, genetically
related to ISA-22 (WRLFMD 2022). These highlight that
similar viruses were detected in East Java, Central Java,
sharing genetic data is important for better understanding
Bangka Belitung, South Kalimantan, Central Kalimantan and
virus evolution to assist FMD control strategies between
West Kalimantan. Interestingly, ISA-22 viruses from West Nusa
affected countries.
Tenggara (ISA/LTE/A06220465/2022 and ISA/LTI/A06220466/
Based on the current FMD situation in neighbouring
2022) were clustered with three ISA-22 viruses from Central
countries of Indonesia where O/ME-SA/Ind-2001e virus is
Java (ISA/Boyolali/A04222562/2022, ISA/Klaten/A04222596/
endemic (Bachanek-Bankowska et al. 2018; Blacksell et al.
2022 and ISA/Semarang/A04222614/2022).
2019; King 2021) and the role of animal movement in the
Sequence analysis on the VP1 protein of ISA-22 viruses
spread of FMD in Asia and other regions (Rweyemamu
revealed that amino acid variations were found at position
et al. 2008; Di Nardo et al. 2011; Smith et al. 2015), the intro-
13 (A/T), 129 (V/A), 142 (T/A), 158 (A/G) and 201 (Y/H)
duction of virus into Indonesia could be predisposed by a
(Table 3). Amino acid changes in VP1 protein of three virus
transboundary ‘illegal’ trade of live animals or FMD-contami-
samples collected from goat (ISA/Jombang/PVTW-J11/2022,
nated materials from endemic areas in Southeast Asia. A
ISA/Mempawah/BBR11/2022 and ISA/HSU/A0522099-5/2022)
study by Bachanek-Bankowska et al. (2018) indicated that
were also found in most of virus samples collected from
although many outbreaks of the Ind-2001d and Ind-2001e
cattle, and no changes were found either at the critical
lineages were due to single independent introductions of
amino acid sites at position 144 (V), 148 (L), 154 (K) and
the virus into new regions, some became established
208 (P), or in the position 145–147 (retained RGD sequence
within different geographic areas, providing additional
motif).
 JOURNAL OF APPLIED ANIMAL RESEARCH 491

Figure 2. Phylogenetic analysis of Foot-and-Mouth Disease (FMD) Subtype O virus collected from disease outbreaks in Indonesia in May 2022. The evolutionary history
was inferred by using the Maximum Likelihood method and the best-fit substitution model for the dataset (HKY+G; Hasegawa-Kishino-Yano with a discrete Gamma
distribution) with 1000 bootstrap replication. The percentage of bootstrap statistical value in which the associated taxa clustered together is shown next to the
branches with values equal or more than 50% are only shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site
(0.05) shown in the left. This analysis involved 96 nucleotide sequences with a total of 639 positions of the VP1 gene of the FMD virus in the final dataset. Evolutionary
analyses were conducted in MEGA X. Three viruses from Cambodia isolated in 2019 (CAM-22) and the ISA-22 viruses (bold-italic taxa name) formed two distinct clusters
within O/ME-SA/Ind-2001e lineage. Three viruses were collected from goats including ISA/Jombang/PVTW-J11/2022, ISA/Mempawah/BBR11/2022 and ISA/HSU/
A0522099-5/2022, while the other twenty-three viruses collected from cattle.
492 E. B. SUSILA ET AL.

Table 2. Average pairwise distance, number of base differences and homology of As shown in Figure 2, Aceh and North Sumatera provinces
ISA-22 viruses to the other virus lineages belonging to O Serotype circulating in are in the same Sumatera Island where they share land
Asia.
borders. Phylogenetic analysis inferred that there might have
Between ISA-22 to the Average
other O-serotype pairwise Average number of Average been an incidence of FMD virus transmission and circulation
lineage distance nucleotide difference homology on the border areas between North Sumatera and Aceh
Ind-2001e 0.047 30.3 95.3% before the transmission event occurred in Java causing out-
Ind-2001e (CAM-19 0.055 35.4 94.5% breaks in domestic livestock and then spread to other areas
Group)
Ind-2001d 0.089 56.8 91.1% in Indonesia. Previous studies identified critical points of
Ind-2001c 0.110 70.5 89.0% disease amplification and transmission, such as holding facili-
Ind-2001b 0.094 60.1 90.6% ties and livestock markets that provide opportunities for exten-
Ind-2001a 0.101 64.5 89.9%
PanAsia2 0.121 77.0 87.9% sive mixing of livestock from different origins that are destined
PanAsia 0.122 77.7 87.8% for different locations (Di Nardo et al. 2011; Smith et al. 2015).
ISA-1 0.191 122.2 80.9% Furthermore, the spread of disease can also be affected by
ISA-2 0.201 128.4 79.9%
Within ISA-22 shared border areas, unofficial cross-border movement, lack
lineage of effective biosecurity and low levels of compliance with
Ind-2001e/ISA-22 0.004 2.4 99.6% animal health regulations (Smith et al. 2015). However, the
There was a total of 639 positions of VP1 gene used for this analysis. lack of extensive data hamper the understanding of how the
The number of base differences per site from averaging over all sequence pairs
between groups is shown. The rate variation among sites was modelled with virus was transmitted in the early stage of FMD cases in Indone-
a gamma distribution (shape parameter = 5). All ambiguous positions were sia. One possibility is that the virus might be transmitted over
removed for each sequence pair (pairwise deletion option). Evolutionary ana- longer distances by infected small ruminants (e.g. sheep and
lyses were conducted in MEGA X.
goats) that show inapparent or mild clinical signs of FMD
(Barnett and Cox 1999) before the disease was reported and
sources for onward transmission. This study also indicated spread in cattle. This hypothesis should be investigated
that the predominant disease occurrences of the Ind-2001e further since FMD viruses seemed able to persist in a sheep
lineage in diverse and distant geographic regions since population with the reproduction ratio only slightly larger
2016 might be linked to increased or altered trading than one (Orsel et al. 2007), yet in a mixed population of
routes as well as movement of people, and such virus- sheep and cattle, sheep play a more limited role in the trans-
specific factors for example replication and/or transmission mission of FMDV than cattle (Bravo de Rueda et al. 2014).
fitness across susceptible host species have played an In summary, after 36 years of maintaining the FMD-free
important role in the establishment of this lineage (Bacha- country, Indonesia reported FMD outbreaks in livestock to
nek-Bankowska et al. 2018). World Organization for Animal Health in May 2022 (WOAH

Table 3. Amino acid changes detected in VP-1 of ISA-22 viruses and critical amino acids found at the VP1 epitope and sites that involved in the adsorption of virus to
host cell.
Amino acid changes
ID Strain name Species origin Accession number 13 129 142 144 145 146 147 148 154 158 201 208
1 ISA/Tamiang/01220759-P4/2022 Cattle ON783873 A V T V R G D L K A Y P
2 ISA/Tamiang/01220759-P10/2022 Cattle ON783874 A V T V R G D L K G H P
3 ISA/Lamongan/PVT-VL6/2022 Cattle ON854936 A A A V R G D L K A H P
4 ISA/Sidoarjo/PVT/2022 Cattle ON854937 A A A V R G D L K A H P
5 ISA/Mojokerto/PVT/2022 Cattle ON854938 A A A V R G D L K A H P
6 ISA/Babel/PVT-AN/2022 Cattle ON854939 A A A V R G D L K A H P
7 ISA/Langkat/A01220761/2022 Cattle ON854934 A V T V R G D L K A H P
8 ISA/DeliSerdang/A01220762/2022 Cattle ON854935 A V T V R G D L K A H P
9 ISA/Probolinggo/PVT/2022 Cattle ON854940 A A A V R G D L K A H P
10 ISA/Malang/PVT/2022 Cattle ON854941 A A A V R G D L K A H P
11 ISA/Boyolali/A04222562/2022 Cattle ON854942 A A A V R G D L K A H P
12 ISA/Pasuruan/A04222583/ 2022 Cattle ON854943 A A A V R G D L K A H P
13 ISA/Pangkal Pinang/ PVT/2022 Cattle ON854944 A A A V R G D L K A H P
14 ISA/LTE/A06220465/2022 Cattle ON854945 T V A V R G D L K A H P
15 ISA/LTI/A06220466/2022 Cattle ON854946 A V A V R G D L K A H P
16 ISA/KWB/A0522106/2022 Cattle ON854948 A A A V R G D L K A H P
17 ISA/Banjarnegara/ A04222582/2022 Cattle ON854949 A A A V R G D L K A H P
18 ISA/Klaten/A04222596/2022 Cattle ON854950 A V A V R G D L K A H P
19 ISA/Gresik/PVT-KBMS/2022 Cattle ON854952 A A A V R G D L K A H P
20 ISA/Purbalingga/ A04222582/2022 Cattle ON854953 A A A V R G D L K A H P
21 ISA/Pemalang/A04222613/2022 Cattle ON854954 A A A V R G D L K A H P
22 ISA/Semarang/ A04222614/2022 Cattle ON854955 A V A V R G D L K A H P
23 ISA/Magetan/A04222620/2022 Cattle ON854956 A A A V R G D L K A H P
24 ISA/Jombang/PVT-J11/2022 Goat ON854951 A A A V R G D L K A H P
25 ISA/Mempawah/BBR11/2022 Goat ON854947 A A A V R G D L K A H P
26 ISA/HSU/A0522099-5/2022 Goat ON854957 A A A V R G D L K A H P
Amino acids at positions 140–160 and 200–213 within VP1 protein contain important epitopes particularly at sites 144 V, 148L, 154 K, and 208P and amino acids at
positions 145–147 comprising RAG residues involved in the absorption of virus to host cell.
ISA/Tamiang/01220759-P4/2022 was selected as reference strain for amino acid sequence analysis. Amino acid changes were indicated in bold characters.
 JOURNAL OF APPLIED ANIMAL RESEARCH 493

2022). Our study reports the detection and identification of the ORCID
FMD O/ME-SA/Ind-2001e virus in Indonesia. This finding pro- Edy Budi Susila http://orcid.org/0009-0002-2464-1243
vided immediate direction for the government of Indonesia Dewi Noor Hidayati http://orcid.org/0000-0001-7584-5065
for FMD control, particularly for vaccination strategy since Sri Handayani Irianingsih http://orcid.org/0000-0003-0652-3518
stamping-out policy was not fully implemented as the Yuni Yupiana http://orcid.org/0000-0002-6478-2879
disease has been spread in multiple areas across islands Hendra Wibawa http://orcid.org/0000-0002-3725-9978
within the country. How the virus was introduced into Indone-
sia remains undetermined; nonetheless, unauthorized move-
ment of live animals from areas where FMD is currently References
endemic is a possible route that requires more investigation.
Bachanek-Bankowska K, Di Nardo A, Wadsworth J, Mioulet V, Pezzoni G,
More advanced studies using whole genome sequencing
Grazioli S, Brocchi E, Kafle SC, Hettiarachchi R, Kumarawadu PL, et al.
data of the FMD virus will provide better insight to infer the 2018. Reconstructing the evolutionary history of pandemic foot-and-
source of virus incursion and to determine the molecular mouth disease viruses: the impact of recombination within the emer-
pattern of virus transmission between areas in Indonesia. ging O/ME-SA/Ind-2001 lineage. Sci Rep. 8(1):14693. doi:10.1038/
s41598-018-32693-8.
Barnett PV, Cox SJ. 1999. The role of small ruminants in the epidemiology
Acknowledgements and transmission of foot-and-mouth disease. Vet J. 158(1):6–13. doi:10.
1053/tvjl.1998.0338.
The authors acknowledge the Director General of Livestock and Animal Blacksell SD, Siengsanan-Lamont J, Kamolsiripichaiporn S, Gleeson LJ,
Health Services (DGLAHS), Ministry of Agriculture of Indonesia in facilitating Windsor PA. 2019. A history of FMD research and control programmes
epidemiological investigations and laboratory confirmation conducted by in Southeast Asia: lessons from the past informing the future.
DGLAHS’ veterinary laboratories. We thank the veterinary laboratory direc- Epidemiol Infect. 147(e171):1–13. doi:10.1017/S0950268819000578.
tors including from DICs, NCVB Surabaya, and NVDAL Bogor and their staff Bravo de Rueda C, de Jong MC, Eble PL, Dekker A. 2014. Estimation of the
for their support on field sampling and laboratory diagnostics for the detec- transmission of foot-and-mouth disease virus from infected sheep to
tion and identification of FMD agents. We also acknowledge the United cattle. Vet Res. 45(1):45. doi:10.1186/1297-9716-45-58.
Nations Food and Agriculture Organization Emergency Centre for Trans- Brito BP, Rodriguez LL, Hammond JM, Pinto J, Perez AM. 2015. Review of the
boundary Animal Diseases, Jakarta, Indonesia, for supporting a diagnostic global distribution of foot-and-mouth disease virus from 2007 to 2014.
algorithm and reagents for molecular identification through rRT-PCR and Transbound Emerg Dis. 64(2):316–332. doi:10.1111/tbed.12373.
DNA sequencing. Di Nardo A, Knowles NJ, Paton DJ. 2011. Combining livestock trade patterns
with phylogenetics to help understand the spread of foot and mouth
disease in sub-Saharan Africa, the Middle East and Southeast Asia. Rev
Disclosure statement Sci Tech. 30(1):63–85. doi:10.20506/rst.30.1.2022.
No potential conflict of interest was reported by the author(s). Grubman MJ, Baxt B. 2004. Foot-and-mouth disease. Clin Microbiol Rev. 17
(2):465–493. doi:10.1128/CMR.17.2.465-493.2004.
Hemadri D, Tosh C, Sanyal A, Venkataramanan R. 2002. Emergence of a new
strain of type O foot-and-mouth disease virus: its phylogenetic and evol-
Ethic statement
utionary relationship with the PanAsia pandemic strain. Virus Genes.
Sample collection was performed as part of routine field out- 25:23–34. doi:10.1023/A:1020165923805.
Jinding C, Mingqiu Z, Hui KH, Leung FC. 2006. Molecular characterization of
break investigations from which all cases described herein
foot-and-mouth disease virus in Hong Kong during 2001–2002. Virus
occurred spontaneously in domestic livestock with no exper- Genes. 32:139–143. doi:10.1007/s11262-005-6869-1.
imental inoculation or treatment of live animals and no King D. 2021. Global foot-and-mouth disease situation: risk and new devel-
animals were euthanized for this study. Therefore, no ethical opment. The 24th SEACFMD National Coordinators Meeting-OIE-Asia.
approval was required for this study as the sample collection https://rr-asia.woah.org/wp-content/uploads/2021/07/01_donald_
king_seacfmd-july-2021.pdf.
was performed using standard diagnostic procedures with no
Knowles NJ, Samuel AR. 2003. Molecular epidemiology of foot-and-mouth
harm to the animals. disease virus. Virus Res. 91(1):65–80. doi:10.1016/S0168-1702(02)00260-
5.
Knowles NJ, Samuel AR, Davies PR, Midgley RJ, Valarche JF. 2005. Pandemic
Author’s contributions strain of foot-and-mouth disease virus serotype O. Emerg Infect Dis. 11
(12):1887–1893. doi:10.3201/eid1112.050908.
Study conception and design: E.B. Susila, H. Wibawa. Drafting
Knowles NJ, Wadsworth J, Bachanek-Bankowska K, King DP. 2016. VP1
the manuscript: E.B. Susila, H. Wibawa, Y. Yupiana. Revised sequencing protocol for foot and mouth disease virus molecular epide-
the manuscript: D.N. Hidayati, H. Wibawa. Virus identification miology. Rev Sci Tech. 35(3):741–755. doi:10.20506/rst.35.3.2565.
through sequencing and phylogenetic analysis: R.S.D. Daulay, Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evol-
E. Andesfha, H. Wibawa. Performed virus detection through lab- utionary genetics analysis across computing platforms. Mol Biol Evol. 35
(6):1547–1549. doi:10.1093/molbev/msy096.
oratory diagnostics and provided outbreak data: S.R.B. Prase-
Le VP, Lee KN, Nguyen T, Kim SM, Cho IS, Khang DD, Hien Nguyen B, Van
tyowati, Wriningati, D.N. Hidayati, S.H. Irianingsih, I.N. Dibia, Quyen D, Park JH. 2012. A rapid molecular strategy for early detection
Faisal, A. Supriyadi. Provided epidemiological data and analysis: and characterization of Vietnamese foot-and-mouth disease virus sero-
Y. Yupiana, M.M. Muharram. Facilitated the study and provided types O, A, and Asia 1. J Virol Methods. 180(1–2):1–6. doi:10.1016/j.
final approval of the manuscript to be published: N. Zainuddin. jviromet.2011.11.028.
Orsel K, Dekker A, Bouma A, Stegeman JA, de Jong MCM. 2007.
Quantification of foot and mouth disease virus excretion and trans-
Data availability statement mission within groups of lambs with and without vaccination. Vaccine.
25(14):2673–2679. doi:10.1016/j.vaccine.2006.11.048.
All sequence data of the FMD-VP1 gene are available in GenBank under Rweyemamu M, Roeder P, Mackay K, Sumption K, Brownlie Y, Leforban J,
accession numbers: ON783873, ON783874, and ON854934 to ON854957. Valarcher J-F, Knowles NJ, Saraiva V. 2008. Epidemiological patterns of
494 E. B. SUSILA ET AL.

foot-and-mouth disease worldwide. Transbound Emerg Dis. 55(1):57–72. WOAH. 2021. Chapter 3.1.8: foot and mouth disease (infection with foot
doi:10.1111/j.1865-1682.2007.01013.x. and mouth disease virus). OIE Terrestrial Manual 2021. https://rr-asia.
Smith P, Lüthi NB, Huachun L, Oo KN, Phonvisay A, Premashthira S, Abila R, woah.org/en/projects/foot-and-mouth-disease-fmd/.
Widders P, Kukreja K, Miller C. 2015. Movement pathways and market WOAH. 2022. Immediate notification of foot-and-mouth disease status from
chains of large ruminants in the Greater Mekong Sub-region. OIE Sub- Indonesia (Reported on 9 May 2022). https://wahis.woah.org/#/report-
Regional Representation for South-East Asia; [accessed 2022 June 6]. info?reportId=53545.
https://rr-asia.woah.org/wp-content/uploads/2019/10/livestock_ WRLFMD. 2022. WHO-FAO FMD reference laboratory report, July-September
movement_pathways_and_markets_in_the_gms__final_.pdf. 2022. Quarterly Report. https://www.wrlfmd.org/ref-lab-reports.