Establishing a Specific qPCR Assay for Detecting Middle Eastern O Serotype Foot-and-Mouth Disease Virus (FMDV)

September 5, 2015 — admin
AttachmentSize
establishing_a_specific_qpcr_assay.pdf188.59 KB
Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 21 qPCR Assay FMDV
Establishing a Specifc qPCR Assay for Detecting Middle Eastern
O Serotype Foot-and-Mouth Disease Virus (FMDV)
Engor, E.,
1
Gelman, B.,
1
Khinitch, E.,
1
Rubinstain, M.,
1
Shwartz, G.,
2
Haegeman, A.
3
and Stram, Y.
1
1
Virology Department, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
2
Agentek Ltd., POB 58008 , Tel Aviv 6158001, Israel.
3
CODA-CERVA Vesicular and Exotic Diseases Veterinary and Agrochemical Research Center Brussels B-1180, Belgium.
*
Corresponding Author: Dr. Yehuda Stram, Virology Department, Kimron Veterinary Institute, P. O. Box 12, Bet Dagan 50250, Israel.
Email: yehudastram@gmail.com
ABSTRACT
Te Middle East is one of the main regions under threat of contracting Foot and Mouth Disease (FMD).
Indeed, Israel and the Palestinian Territory sufered in the last years from several outbreaks. Te FMD viruses
responsible for the Middle Eastern outbreaks were predominantly associated with O serotype. Phylogenetic
data has indicated that viruses are introduced to the area from diferent regions, ranging from the Arabian
peninsula to the Indian sub-continent. Accurate and rapid identifcation of the infectious pathogen is essential
in endemic areas such as the Middle-East to enable a proper response to combat the disease. In recent years
the use of qPCR has become a common practice in the diagnosis of FMDV. A qRT-PCR assay has been
developed permitting the discrimination between past and recent Middle Eastern FMDV O type, and the
other 6 FMDV serotypes. Moreover, the developed assay, beside, the ability to detect existing strains will
probably be able to identify new infecting strains of virus.
Keywords: Foot and Mouth Disease; Middle East; O Serotype; qRT-PCR Assay; VP1 Sequence;
Biosecurity
INTRODUCTION
Foot-and-mouth disease virus (FMDV) is one of the most
devastating viruses that afect cloven-hoofed animals. Te
capacity of the virus to spread and to modify its antigenic
identity makes it a signifcant threat to the beef and dairy
industries in many countries. One of the FMD-endemic re-
gions of the world is the Middle East, where FMD outbreaks
occur almost each year (1). Te virus belongs to the genus
Aphthovirus in the Picornaviridae family. Te virus genome is
an 8.3-kb single stranded RNA in the plus orientation car-
rying a poly-A tract at its 3’ end and a viral genome protein
(VPg) at its 5’ end (2-4). Tere are seven diferent virus sero-
types that do not mutually cross-protect, and each comprises
of numerous subtypes; about 80 in total (5). Te large number
of subtypes results from the high rate of mutation, especially
in the VP1 gene (6-9). Te VP1 gene encodes a structural
protein exposed on the surface of the virion which carries the
major antigenic sites for the immunological identity of the
virus (10-12). Te traditional means of protection is vaccina-
tion, which greatly reduces the occurrence of the disease (5).
Nevertheless, there are hundreds of outbreaks in Asia, Africa,
South America and Eastern Europe each year, whereas North
America, Australia and Western Europe are virus-free regions
and domestic animals there are not vaccinated (13, 14).
Te Middle East, including Israel, is one of the main areas
where FMDV resides: there are outbreaks almost every year,
some of them major with large numbers of infected farms
across the country. In recent years the O-type virus has been
the prominent type responsible for outbreaks in the Middle
East, and throughout the years a large collection of the vi-
ruses have been accumulated, enabling the establishment
of an Israeli database. Te fact that the majority of FMD
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Engor, E. 22
outbreaks were of O serotype prompted the establishment of
a qPCR protocol utilizing VP1 sequences as templates for the
specifc detection of O serotype viruses that were responsible
for past outbreaks of FMDV in Israel (15).
Te aim of this publication was to describe a recently de-
veloped assay based on degenerate primers and 2 probes with
diferent fuorophores resulting in a superior probability of
identifying a current infectious strain and/or future serotype
O viruses entering the Middle East.
MATERIAL AND METHODS
RNA extraction and qPCR
RNA was extracted using Viral Gene-spin (Intron
Biotechnology, South Korea) according to the manufacturer’s
protocol.
Primes and Probes
Primers (Sigma-Aldrich, Israel) and probes (Bioresearch
Technologies, USA) sequences are listed in Table 1.
qPCR
A single step RT-qPCR was performed by using qScript
XLT mix (Quanta BioSciences, MD, USA) according to
the manufacturer’s protocol. qPCR systems (primers and
probes) were designed and prepared by Biosearch technolo-
gies (Biosearch technologies, CA, USA).
Bioinformatics
Multiple homology analysis was performed using ClustalW2
(http://www.ebi.ac.uk/Tools/msa/clustalw2/). EMBOSS set
of programs were used for other bioinformatics activities.
RESULTS AND DISCUSSION
In order to institute a qPCR able to identify specifcally sero-
type O FMD, full FMDV O viral genome sequences from the
Middle East and neighboring countries were used to search for
highly conserved sequences suitablefor Taqman assay (Table
1). To assess the ability of the assay to identify FMDV of O
serotype, isolates from 1982-2014 were examined. All isolates
were successfully detected except for Neve Ur 2010 and Tira
2014 isolates (Table 2).
To reveal whether genomic changes were the cause of the
failure to react with Neve Ur and Tira isolates, a ClustalW2
analysis was carried out with Israeli VP1 sequences of recent
isolates together with the primers and probe used (Figure
1). Tis analysis clearly showed an important degree of vari-
ability in the probe binding site and to a lesser degree in
both primer binding sites resulting in critical changes had to
be made particularly in the probe and less so in both which
primers sequences. As for the probe, the C at position 5 was
changed to T, the A at position 9 was changed to A/G (R),
the C at position 14 was changed to C/T (Y), and the C at
position 17 was changed to T. Similarly, the forward primer
was adapted by changing the G at position 12 to A (since it is
present in all tested sequences except for /IRN/18/2010) and
C at position 15 was changed to Y. Tere were fewer changes
made in the reverse primer: T instead of G at position 8, and
C instead of G at position 11 (Table 1).
Te two assays were utilized together in a multiplex for-
mat for examining their ability to identify all the samples.
Te method was examined with all Israeli isolates from
1982-2014, and was shown that all samples including Neve
Ur 2010 and Tira 2014, which were undetectable by the frst
assay (Table 2), were now detected.
Table 1: In-house designed primers and probes used in this work
First qPCR system Sequence Position in acc. KM921827
Forward primer GTGGCAGTGAAG*CAC*GAGG 232
probe FAM-TGGAC*AACA*CCACC*AAC*CCAACA*G-BHQ 293
Reverse primer GTAGCCAAC*AC*CGGTGTG 365
Second qPCR system
Forward primer GTGGCAGTGAAA^CAY^GAGG 232
probe VIC-TGGAT^AACR^CCACY^AAT^CCAACG^G-BHQ 293
Reverse primer GTAGCCAAG^ACA^CGGTGTG 365
* – indicate nucleotides to be changed. ^ – indicate changed nucleotides.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 23 qPCR Assay FMDV
O/ISR/1/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/ISR/2/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/ISR/3/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
Madgel-Shams CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 299
PAK10-2006 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 300
PAK14-06 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
PAK08-06 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
JOR-06 CGCAGATCTAGAGGTGGCAGTGAAACATGAGGGGAGCCTTACCTGGGTCCCGAATGGGGC 278
BHU15-03 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
NEP-03 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
BHU49-03 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
AFG-04 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
AFG-03 CGCAGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/IRN/49/2009 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
Jerico-2013-11 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTCACCTGGGTCCCGAATGGGGC 278
O/PAT/13/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTCACCTGGGTCCCGAATGGGGC 278
O/PAT/14/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTCACCTGGGTCCCGAATGGGGC 278
O/TUR/12/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/TUR/38/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/TUR/37/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/TUR/27/2013 CGCAGATTTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGC 278
O/IRN/18/2010 CGCAGATCTAGAGGTGGCAGTGAAACATGAAGGGAACCTTACCTGGGTACCCAATGGGGC 278
Forward primer GTGGCAGTGAAGCACGAGG
***** ***** ** * *
New GTGGCAGTGAAACAYGAGG
O/ISR/1/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/ISR/2/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/ISR/3/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
Madgel-Shams GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 359
PAK10-2006 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAGGCACCGCTCAC 360
PAK14-06 GCCCGAGACAGCGTTGGATAACACCACTAACCCAACGGCTTACCACAAGGCACCGCTCAC 338
PAK08-06 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAGGCACCGCTCAC 338
JOR-06 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAAGCACCGCTCAC 338
BHU15-03 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAGGCACCGCTCAC 338
NEP-03 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAGGCACCGCTCAC 338
BHU49-03 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAGGCACCGCTCAC 338
AFG-04 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAGGCACCGCTCAC 338
AFG-03 GCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCCTACCACAAGGCACCGCTCAC 338
O/IRN/49/2009 GCCCGAGACAGCGTTGGATAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
Jerico-2013-11 GCCCGAGACAGCGTTGGACAACGCCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/PAT/13/2013 GCCCGAGACAGCGTTGGACAACGCCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/PAT/14/2013 GCCCGAGACAGCGTTGGACAACGCCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/TUR/12/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/TUR/38/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/TUR/37/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/TUR/27/2013 GCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCACTCAC 338
O/IRN/18/2010 GCCTGAGAAGGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCGCTCAC 338
probe TGGACAACACCACCAACCCAACAG
**** *** **** ** ***** *
New TGGATAACRCCACYAATCCAACGG
rfO/ISR/1/2013 CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/ISR/2/2013 CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/ISR/3/2013 CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
Madgel-Shams CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 419
PAK10-2006 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAA 420
PAK14-06 CCGACTTGCACTGCCTTACACGGCACCACACCGAGTCTTGGCTACCGTATACAACGGGAA 398
PAK08-06 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAA 398
JOR-06 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAA 398
BHU15-03 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACTGTTTACAACGGGAA 398
NEP-03 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACTGTTTACAACGGGAA 398
BHU49-03 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACTGTTTACAACGGGAA 398
AFG-04 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACTGTTTACAACGGGAA 398
AFG-03 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACTGTTTACAACGGGAA 398
O/IRN/49/2009 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
Jerico-2013-11 CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/PAT/13/2013 CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/PAT/14/2013 CCGACTTGCCCTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/TUR/12/2013 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/TUR/38/2013 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/TUR/37/2013 TCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/TUR/27/2013 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTATACAACGGGAG 398
O/IRN/18/2010 CCGACTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACCGTGTACAACGGGAA 398
reverse primer CACACCGCGTGTTGGCTAC
******* ** ********
New CACACCGTGTCTTGGCTAC
Figure 1: ClustalW2 analysis of primers, Probes, and VP1 sequences of recent Israelis isolates. * – indicates the nucleotide changes in the new
detection system. Accession no. are as published (Stram et al., 2011: Stram et al., 2015, submitted for publication)
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Engor, E. 24
To further validate the method, a comparison was made
with a non-specifc qPCR assay, able to detect any FMD
virus regardless of theserotype, based on the conserved 3D
region (16). It was noticed that the newly developed assay was
in full agreement with the FMDV non-specifc qPCR assay.
Furthermore, it was observed that several samples the
cycle threshold (C
T
) of both assays (O specifc and non-
specifc) were highly similar. Dalton 1982 with C
T
of 24.54
and 25.59 , Neve Ur 2008 with C
T
21.72 and 21.77 Nature
2008 with C
T
of 18.58 and 17.71, respectively, were found
to be similar (Table 2), although other samples difered in
their C
T
values. Tis validated the new assay as suitable to
be used as a diagnosis tool for the isolates with the potential
for detecting new incoming O serotype viruses.
To assess the specifcity of the new method all seven
FMDV serotypes (Table 3) were utilize in the assay. All
serotypes except for O were negative. It is worth noticing
that isolate C NEP 1/94 showed C
T
value of 36, which is
considered negative or at least signifcantly non-positive.
In order to demonstrate the practicality of the test, a
tongue epithelial sample taken during an outbreak in the
Palestinian Territories (Dura-Daharya 2014) was tested
utilizing the recently established procedure in addition to
the general qPCR assay. It was evident that both tests could
detect and identify the virus responsible for the disease out-
break with C
T
of 25.5 with the general FMDV test and C
T
of 28.3 with FMDV O test (Table 3).
Like any other qPCR assays, the technique is highly
sensitive to the template mutations particularly in the probe
sequences. It was suggested that major outbreaks in the
Middle East, including Israel, result from a newly arrived
virus (15). Tus, there is always a chance, as remote as it is,
of a virus with sufcient nucleotide changes in the primers
and probe region arriving, which will escape detection.To
illustrate the probability of such an occurrence, a system able
to detect Middle Eastern O serotype (17) was examined by
performing homology analysis using the probe and primers
Table 2: Comparison between the frst, the improved O specifc and the non-specifc FMDV qRT-PCR assays. = Te FMDV O specifc assays
were performed as multiplex reactions
Sample Name
First O Specifc assay C
T
O Specifc assay C
T
general FMDV assay C
T
Dalton1982 20.22 24.54 25.49
Gshur 2000 13.29 17.65 NT
Never Ur 2008 26.95 21.72 21.77
Natur 2008 23.88 18.58 17.75
Never Ur 2010 UD 17.75 NT
Manisa 2011 17.01 20.98 13.89
Gshur 2013 15.73 19.90 16.29
Jerico 2013 19.98 15.35 NT
Tira 2014 UD 14.28 NT
Dura-Daharya 2014 - 28.12 25.2
Negative UD UD UD
A ISR 2009
UD
UD UD
UD – undetected. NT – not tested.
Table 3: Specifcity test of the new qPCR assay. Samples of all seven
FMDV serotypes were tested by the newly developed qRT-PCR
Sample Name C
T
A – Iraq 06 UD
A – Egp 13 UD
A – Kenya 11 UD
Asia 1 - Shamir UD
C SRL 1/84 UD
C SAU 1/84 UD
C BHU 2 /94 UD
C PHI 11/89 UD
C NEP 1/94 36.05
SAT-1 Bot UD
SAT-2 Zim UD
SAT-3 Zim UD
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 25 qPCR Assay FMDV
with the Israeli VP1 database. It was shown that in the 2013-
14 isolates responsible for several outbreaks in the Palestinian
Territories the A located at the 3’ of the primer was changed
to G. It was considered that this change could possibly ham-
per the ability of the assay to detect these viruses.
In summary, this work represents the development of
a qRT-PCR assay that enables the detection of FMDV
O serotype viruses found in past in the Middle East and
provides a highlikelihood that it will be able to detect future
introductions of O serotype viruses. It was already reported
that the Middle East is a sensitive region for constant intro-
duction of FMDV and particularly of O serotype. Terefore,
it is paramount to have an assay available that is suitable for
the task of detecting viruses residing in the area with the
ability to identify future and new virus introductions, thus
making it valuable to Middle Eastern laboratories engaged
with FMDV.
REFERENCES
1. Stram, Y., Chai, D., Fawzy, H.E.-D., Molad, T., Meiri, N., Van-
Ham, M., El-Kilani, S., Fahamy, F., Moussa, A.A.M. and Yadin,
H.: Molecular epidemiology of foot-and-mouth disease (FMD) in
Israel in 1994 and in other Middle-Eastern countries in the years
1992-1994. Arch. Virol., 140: 1791-1798, 1995.
2. Carroll, A.R., Rowlands, D.J. and Clark. B.E.: Te complete nu-
cleotide sequence of the RNA for the primary translation product
of foot and mouth disease virus. Nucl. Acid. Res. 12: 2461-2472,
1985.
3. Fross, S., Strebel, K., Beck, E. and Scaller, H.: Nucleotide sequence
and genomic organization of Foot and Mouth Disease Virus. Nucl.
Acids Res. 12: 6587-6601, 1984.
4. Li, D., Shang, Y.J., Liu, Z.X., Liu, X.T. and Cai, X.P.: Compari-
sons of the complete genomes of two Chinese isolates of a recent
foot-and-mouth disease type Asia 1 virus. Arch. Virol. 2007, 152,
1699-1708.
5. Mason, P.W., Grubman, M.J. and Baxt, B.: Molecular basis of
pathogenesis of FMDV. Virus Res. 91: 9-32, 2003.
6. Carrillo, C., Tulman, E.R., Delhon, G., Lu, Z., Carreno, A., Vag-
nozzi, A., Kutish, G.F. and Rock, D.L.: Comparative genomics
of foot-and-mouth disease virus. J. Virol. 79: 6487-6504, 2005.
7. Domingo, E., Mateu, M.G., Martinez, M.A., Dopazo, J., Moya,
A. and Sobrino, F.: Genetic variability and antigenic diversity of
foot-and-mouth disease virus. In: Kurstak, E., Marusyk, R.G.,
Murphy, E.A., Van Regermortel, M.H.V. (Eds.), Virus Variability,
Epidemiology and Control, vol. 2. Plenum Publishing Corpora-
tion, New York, pp. 233-266, 1990.
8. Dopozo, J., Sobrino, F., Palma, E.L., Domingo, E. and Moya, A.:
Gene encoding capsid protein VP1 of foot and mouth disease
virus: A quasispecies model of molecular evolution. Proc. Natl.
Acad. Sci. USA. 85: 6811-6815, 1988.
9. Samuel, A.R., Knowles, N.J. and Mackay, D.K.J.: Genetic analysis
of type O viruses responsible for epidemics of foot-and-mouth
disease in North Africa. Epidemiol. Infect. 122: 529-538, 1999.
10. Barnett, P., V., Ouldridge, E.J., Rowlands, D.J., Brown, F. and
Parry, N.R.: Neutralizing epitopes of type O foot-and-mouth
disease virus. I. Identifcation and characterization of three
functionally independent, conformational sites. J. Gen. Virol. 70:
1483-1491, 1989.
11. Kitson, J.D., McCahon, D. and Belsham, G.J.: Sequence analysis of
monoclonal antibody resistant mutants of type O foot and mouth
disease virus: evidence for the involvement of the three surface
exposed capsid proteins in four antigenic sites. Virol. 179: 26-34,
1990.
12. Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jack-
son, T., King, A., Lea, S., Lewis, R., Newman, J., Parry, N.,
Rowlands, D., Stuart, D. and Fry, E.: Structure of a major im-
munogenic site on foot-and-mouth disease virus. Nature. 362:
566-568, 1993.
13. Valdazo-González, B., Timina, A., Scherbakov, A., Abdul-Ha-
mid, N.F., Knowles, N.J. and King, D.P.: Multiple introductions
of serotype O foot-and-mouth disease viruses into East Asia
in 2010-2011. Vet Res. 44: 76. doi: 10.1186/1297-9716-44-76,
2013/.
14. Jamal, S.M. and Belsham, G.J.: Foot-and-mouth disease: past, pre-
sent and future. Vet Res. 44: 116. doi: 10.1186/1297-9716-44-116.
15. Stram, Y., Engel, O., Rubinstein, M., Kuznetzova, L., Balaish, M.,
Yadin, H., Istumin, S. and and Gelman, B.: Multiple invasions of
O1 FMDV serotype into Israel revealed by genetic analysis of
VP1 genes of Israeli’s isolates from 1989 to 2007. Vet. Microbiol.
147: 398-402, 2011.
16. Abd El Wahed, A., El-Deeb, A., El-Toloth, M., Abd El Kader,
H., Ahmed, A., Hassan, S., Hofmann, B., Haas, B., Shalaby, M.
A., Hufert, F.T. and Weidmann, M.A.: Portable reverse transcrip-
tion recombinase polymerase amplifcation assay for rapid detec-
tion of foot-and-mouth disease virus. PLoS One. 8:e71642. doi:
10.1371/journal.pone.0071642, 2013.
17. Reid, S.M., Mioulet, V., Knowles, N.J., Shirazi, N., Belsham, G. J.
and King, D.P.: Development of tailored real-time RT-PCR assays
for the detection and diferentiation of serotype O, A and Asia-1
foot-and-mouth disease virus lineages circulating in the Middle
East. J. Virol. Methods. 207: 146-153, 2014.
Research Articles

Published under a Creative Commons License By attribution, non-commercial