Emergence of antigenic variants within serotype A FMDV in the Middle East with antigenically critical amino acid substitutions.

A new immunologically distinct strain (A-Iran-05) of foot-and-mouth disease virus serotype A emerged in the Middle East in 2003 that replaced the previously circulating strains (A-Iran-96 and A-Iran-99) in the region. This resulted in introduction of a new vaccine of this strain (A/TUR/2006) in 2006. Though this vaccine strain has been predominantly used to control FMD in the region, recent viruses isolated in 2012 and 2013 have shown antigenic drift and a poor match with it. In this study, we report the antigenic matching results and capsid sequence data of currently circulating viruses belonging to the SIS-10 and SIS-12 sub-lineages of A-Iran-05 (isolated in 2012 and 2013), highlighting the inadequacy of the currently used serotype A vaccines. Implications of these results in the context of FMD control in the Middle East are discussed.

Introduction

Foot-and-mouth disease (FMD) affects both domestic and wild cloven-hoofed animals and remains a globally important livestock animal disease. It has devastating economic implications whenever outbreaks occur in disease free countries as in 2001 in the UK and in 2011 in Japan. In developing countries where the disease is endemic, as well as ongoing direct losses, the export of live animals and animal products is compromised. Vaccination is an important tool for FMD control. The causative agent, FMD virus (FMDV) is a single-stranded, positive-sense RNA virus, the prototype virus of the genus Aphthovirus, family Picornaviridae. Currently, six immunologically distinct serotypes (O, A, Asia 1, South African Territory 1, 2 and 3), each with multiple subtypes are found in the world; no outbreaks due to the seventh serotype “C” have been reported since 2004.

More than 60% of outbreaks worldwide are caused by serotype O, followed by serotype A (http://www.wrlfmd.org). Serotype A viruses are present on all continents where FMD is reported, and are antigenically diverse with as many as 32 distinct subtypes, often with incomplete cross-protection between them [1]. Three serotypes, O, A and Asia 1 have predominated in the Middle East (ME) in recent years, with incursions of SAT 2 viruses in Bahrain and Palestine in 2012 (http://www.wrlfmd.org), probably because of trade links with African countries. Serotype O outbreaks in the region are mainly controlled by O Manisa and O Pan-Asia 2 vaccines which provide broad-spectrum protection. However, the situation with serotype A outbreaks is different because of periodic emergence of antigenically distinct variants, necessitating development of new vaccine strains, typically every 5–10 years. Following the emergence of the antigenically distinct A-Iran-05 genotype in the ME, a new vaccine strain (v/s), A/TUR/2006 was developed by the FMD vaccine manufacturing companies [2] and has been routinely used for FMD control in the region successfully since 2006. However, some recent isolates have exhibited poor cross-reactivity in in vitro serological matching tests, indicating lower expected protection. In a previous study, we reported the inadequacy of serotype A vaccines against viruses from the BAR-08 and ARD-07 sub-lineages and identified surface exposed capsid amino acid residue differences that could underlie the miss-matches [3]. In this study, we report similar studies with viruses from the SIS-10 and SIS-12 sub-lineages (isolated in 2012 and 2013 from Iran and Turkey) also showing low cross-reactivity with three post-vaccinal sera, raised against two old (A22/IRQ/24/64 and A/TUR/2006) and one new (A/IRN/78/2009) candidate vaccine strain. These results extend our previous findings, highlighting the inadequacy of the currently used serotype A v/s for use in FMD control programmes in the region and also identifying further capsid amino acid (aa) residues of likely antigenic significance.

Materials and methods

Cells and viruses: Eighteen serotype A viruses from the ME submitted to the Food and Agriculture Organisation's World Reference Laboratory for FMD (WRLFMD) at Pirbright were used in this study (Table 1). Two are the v/s A22/IRQ/24/64 (A22/Iraq) and A/TUR/2006 that were originally isolated in Iraq and Turkey, in 1964 and 2006 respectively; the sixteen other viruses were isolated over a five year period between 2009 and 2013 (Table 1). With the exception of a single isolate of unknown origin, all samples were derived from cattle epithelial tissues, and were initially grown in primary bovine thyroid cells (BTY) with subsequent passage in either BHK-21 or IB-RS2 cells. Stocks of virus were prepared by infecting IB-RS2 cell monolayers and were stored as clarified tissue culture harvest material at −70 °C until required.

Table 1

List of serotype A viruses used in this study. ND: not designated; NK: not known; N/A: not applicable. The P1 sequences have been submitted to Gene Bank and awaiting accession numbers.

No.	Name	Topotype	Strain	Sub-lineage	Country, region	Host	Date collected	Accession No.
1	A22/IRQ/24/64	ASIA	A22	ND	Mosul, Iraq	Bovine	1964	Upadhyaya et al. [3]
2	A/TUR/2006	ASIA	A-Iran-2005	ND	Eskisehir, Turkey	Bovine	23/06/2006	Upadhyaya et al. [3]
3	A/IRN/78/2009	ASIA	A-Iran-2005	FAR-09	Fars, Iran	NK	04/11/2009	Upadhyaya et al. [3]
4	A/IRN/15/2012	ASIA	A-Iran-2005	SIS-12	Zahedan, Iran	Bovine	24/06/2012	This study
5	A/IRN/22/2012	ASIA	A-Iran-2005	SIS-10	Qom, Iran	Bovine	09/07/2012	This study
6	A/IRN/24/2012	ASIA	A-Iran-2005	SIS-10	Tehran, Iran	Bovine	09/10/2012	This study
7	A/IRN/25/2012	ASIA	A-Iran-2005	SIS-12	Tehran, Iran	Bovine	25/10/2012	This study
8	A/IRN/30/2012	ASIA	A-Iran-2005	SIS-12	Mazandaran, Iran	Bovine	05/11/2012	This study
9	A/IRN/32/2012	ASIA	A-Iran-2005	SIS-10	Yazd, Iran	Bovine	08/11/2012	This study
10	A/IRN/33/2012	ASIA	A-Iran-2005	SIS-12	Esfahan, Iran	Bovine	17/11/2012	This study
11	A/IRN/35/2012	ASIA	A-Iran-2005	SIS-12	Qom, Iran	Bovine	18/12/2012	This study
12	A/IRN/37/2012a	ASIA	A-Iran-2005	SIS-10	Alborz, Iran	Bovine	24/12/2012	N/A
13	A/TUR/05/2012	ASIA	A-Iran-2005	SIS-10	Cavuslar, Turkey	Bovine	11/01/2012	This study
14	A/IRN/01/2013	ASIA	A-Iran-2005	SIS-10	Tehran, Iran	Bovine	01/01/2013	This study
15	A/IRN/04/2013	ASIA	A-Iran-2005	SIS-10	Alborz, Iran	Bovine	01/01/2013	This study
16	A/IRN/06/2013	ASIA	A-Iran-2005	SIS-10	East Azerbaijan, Iran	Bovine	01/01/2013	This study
17	A/IRN/07/2013	ASIA	A-Iran-2005	SIS-10	Ardabil, Iran	Bovine	01/01/2013	This study
18	A/IRN/10/2013a	ASIA	A-Iran-2005	SIS-10	Tehran, Iran	Bovine	01/01/2013	N/A

P1 sequence of these viruses could not be generated in this study.

Polyclonal serum: Pooled bovine post-vaccination sera (BVS) raised against A22/Iraq and A/TUR/2006 v/s [3] were used in this study. In addition, BVS were raised against two new field isolates, A/IRN/78/2009 isolated from Iran in 2009 (sub-lineage: FAR-09) and A/IRN/07/2013 (sub-lineage: SIS-10), essentially as described earlier (3). These last two isolates do not serologically cross-react with either of the established serotype A v/s (A22/Iraq and A/TUR/2006) used in the region. Briefly, five cattle per v/s were immunised with inactivated, purified 146S FMD virus particles in ISA-206 adjuvant. Bulk blood was collected on either 21or 28 days post-vaccination from the immunised animals for preparation of sera. For each antigen, a pool of sera from five animals was used in the serological tests. The A/IRN/78/2009 and A/IRN/07/2013 anti-sera exhibited equivalent titres (log10 2.39 and 2.25, respectively) by virus neutralisation test (VNT) using homologous virus.

Two-dimensional micro neutralisation assay (2D-VNT): The 2D-VNT was carried out using pooled post-vaccination sera following established methodology [4]. Antibody titres were calculated from regression data as the log10 reciprocal antibody dilution required for 50% neutralisation of 100 tissue culture infective units of virus (log10 SN50/100 TCID50). The antigenic relationship of viruses based on their neutralisation by antibodies is given by the ratio: ‘r1’ = neutralising antibody titre against the heterologous virus/neutralising antibody titre against the homologous virus. The significance of differences in the values of ‘r1’ obtained by the polyclonal antiserum was evaluated according to standard criteria [5].

Nucleotide (nt) sequencing and analysis of the sequence data: The sequences of the entire capsid coding region (P1) of SIS-10 AND 12 viruses were generated in order to compare their deduced amino acid (aa) compositions. RNA extraction from the cell culture grown viruses, reverse transcription (RT), polymerase chain reaction (PCR) and sequencing was performed as described previously [3]. The computer analysis of viral capsid sequences were performed by DNASTAR Lasergene 10 EN. The complete P1 sequence of the viruses belonging to the A-Iran-05 strain were aligned and used to construct distance matrices using the Kimura 2-parameter nucleotide substitution model [6] as implemented in the programme MEGA 6.0 [7]. The aligned P1 sequences were used to construct phylogenetic trees using MEGA 6.0.

Data analysis: The data analysis was carried out using Minitab 7.0 software.

Results and discussion

The A-Iran-05 viruses were first detected in Iran in 2003 [2] and rapidly spread to neighbouring countries in the Middle East causing serious problems in Iran in 2005, spreading very quickly to the neighbouring countries. Subsequently, they produced various sub-lineages. Most sub-lineages gradually died out, but a few have persisted. In the last six years, seven sub-lineages (AFG-07, BAR-08, FAR-09, SIS-10, HER-10, FAR-11 and SIS-12) have been regularly detected in the region (Table 2). In addition, other sub-lineages like PAK-09, QAZ-11 and ESF-10 are also occasionally detected (http://www.wrlfmd.org). In this study, we have focussed mainly on two sub-lineages of A-Iran-05, namely SIS-10 and SIS-12 which are currently circulating in the region. SIS-10 viruses were first detected from cattle in an outbreak in Sistan and Baluchestan province in Iran in April 2010. Gradually, there was spread to other provinces in the west, such as Chaharmahal and Bakhtaran (January 2011), in central Iran such as Yazd (January 2011), and to the north as far as Tehran and Qom by January 2011. The lineage continued to circulate in central Iran (Esphan – October 2011), West Azerbaijan (November 2011), Qom (July 2012), Tehran (October 2012), Khuzestan and Yazd Province (November 2012) and Alborz province near Tehran (December 2012 to January 2013), Tehran and East Azerbaijan Province (January 2013), Ardabil Province (January 2013). SIS-10 viruses spread to Turkey in the later part of 2011 and have been circulating in Anatolia, Black sea and Marmara regions (February–March 2013). Viruses of this lineage also spread to Russia and were detected in Garnukha, Urupsky, and Karachay-Cherkess in mid-2013. SIS-12 viruses were first detected in Zahedan, Sistan and Baluchestan province in Iran from cattle in June 2012. The viruses spread to Tehran by October 2012, and were still circulating in Sistan and Baluchestan province, Mazandaran Province, Esphan province, and Qom province in 2012. These viruses were first detected in Pakistan in the Gujrat district in Punjab province in September 2012 and in Islamabad in November 2012.

Table 2

List of sub-lineages of A-Iran-05 viruses detected in the Middle East and neighbouring countries during 2009–2015. AFG: Afghanistan, IRN: Iran, IRQ: Iraq, PAK: Pakistan, TUR: Turkey; –: not detected.

Name of the country/sub-lineagesa	AFG	IRN	IRQ	PAK	TUR
AFG-07b	2009– 11	2009–12	2010	2009–11	2010–11
BAR-08b	–	2009–10	2009	2009	2009
FAR-09	–	2009–10, 2013	–	2014–15	–
SIS-10b	–	2010–14	2013	–	2011–14
HER-10b	2011	2011–12	–	2012	–
FAR-11	2013	2011, 2013–14	–	2011–15	–
SIS-12	–	2012–13	–	2012–13	–

Other sub-lineages detected in only one country are as follows: ARD-07 in Turkey (2008–2010), ESF-10 in Iran (2010), EZM-07 in Turkey (2008–2009), PAK-09 in Pakistan (2009), SIN-08 in Pakistan (2008) and QAZ-11 in Iran (2011). AFG-07 was detected in Bahrain (2009); BAR-08 in Libya (2009), Egypt (2010–2011, 2013), Israel (2009), Lebanon (2009), Palestine (2009), and Kuwait (2009); HER-10 in Kazakhstan (2012), Bahrain (2011) and Kyrgyzstan (2011), and SIS-10 in Russia (2013).

Only sub-lineages detected in more than one country are listed in this table.

Sub-lineages that were detected in other Asian or African countries.

Serological cross-reaction between recent field isolates and vaccine strains

The results of the 2D-VNT test using 15 SIS-10/12 viruses from the ME are presented in Fig. 1. The three v/s did not cross-react with each other indicating they are antigenically distinct. The recent field viruses isolated in 2012 and 2013 exhibited very poor cross-reactivity with A22/Iraq BVS (Fig. 1) indicating the v/s is not suitable for use in the region for FMD control. Similarly, the viruses exhibited poor cross-reactivity with A/TUR/2006 BVS except A/IRN/15/2012, A/IRN/25/2012, A/IRN/35/2012 and A/IRN/10/2013 (Fig. 1). The reactivity pattern of the recent field isolates with A/IRN/78/2009 (belonging to FAR-09 sub-lineage) BVS was very similar to A/TUR/2006 v/s exhibiting moderate cross-reactivity only with two isolates (A/IRN/25/2012 and A/IRN/35/2012) employed in this study (Fig. 1). This indicates that the recent viruses of SIS-10 or SIS-12 sub-lineage, mainly circulating in Iran, Turkey, Pakistan and Russia may require a new vaccine to control FMD outbreaks in the region although a highly potent vaccine can protect against viruses showing low r1-values in some cases [8]. Therefore in vivochallenge studies could confirm these findings.

Fig. 1

Antigenic relationship (r1) values of SIS-10 and SIS-12 viruses. The horizontal dotted line indicates the cut-off value of 0.3, above which the vaccine is considered to be a good match.

Full capsid sequence analysis

The full capsid sequence of 13 SIS-10 and 12 viruses were generated in this study. The capsid sequence of the two v/s and A/IRN/78/2009 were reported in a previous study [3]. The capsid sequences of the other two isolates (A/IRN/37/2012 and A/IRN/10/2013) used in this study could not be generated either because of problems in amplifying the capsid encoding sequences or having ambiguities at more than 15 nt positions that could not be resolved even after repeated attempts. All of the sequences (n = 13) were 2205 nucleotide (nt) long. VP4 was found to be completely conserved at amino acid (aa) level while VP1 was the most variable (data not shown). For phylogenetic analysis the A-Iran-05 virus (2009 onwards) capsid sequences (n = 28) previously generated in our laboratory [3] or available in GenBank (n = 4) were also included. Phylogenetic analysis of the capsid sequences revealed all these viruses belong to the Asia topotype within serotype A FMDV and grouped with viruses belonging to the A-Iran-05 genotype (Fig. 2A). The phylogenetic tree based on VP1 sequence was broadly in agreement with the tree generated using the full capsid sequence (data not shown).

Fig. 2

(A) Phylogenetic tree of the SIS-10 and SIS-12 viruses. The sub-lineages defined by WRLFMD on the basis of VP1 sequences are shown in the figure. The vaccines (existing and putative strains) used in this study are underlined. (B) Capsid amino acid substitution in the SIS-10 and SIS-12 viruses compared to A22/IRQ/24/64, A/TUR/2006 and A/IRN/78/2009 v/s. The residues changed from the v/s are boxed. The grey boxes indicate substitutions in ten out of 15 viruses. The residue positions potentially antigenically important for the antigenic drift from A/TUR/2006 v/s are shown in bold and underlined. (C and D) 3-D structure of Type A10 protomer showing capsid amino acid residues identified in this study; VP1 96 (red), VP2 191 (green), VP3 65 (blue). Residues identified in 10 out of 15 viruses: VP1 – magenta, VP3 – yellow. Residues of potential antigenic significance in case of A/TUR/2006 v/s: VP1 – raspberry, VP2 – orange, VP3 – cyan, VP1 141 not on structure. (C) – cartoon, (D) – external surface. (E) A10 structure with two protomers showing proximity of C-terminal residues of VP1 in an adjacent protomer (mesh). Colour code is same as in (D). Red circles indicate antigenic site 1 residues (VP1 198, 201 and 204) of an adjacent protomer. The white circles indicate the three antigenic sites.

The capsid sequence of all the available SIS-10 and -12 viruses (n = 15) [13 generated in this study along with the two SIS-10 viruses (A/IRN/125/2010 and A/TUR/43/2010) generated in our previous study] were analysed. VP4 was found to be completely conserved at aa level except an aspargine (N) to serine (S) change at position 61 in A/TUR/43/2010 virus. All these viruses exhibited significantly higher variation at nt level when compared to A22/Iraq capsid sequence than the other two vaccines used in this study (14.0–15.4% variation from A22/Iraq compared to 5.0–7.4% variation from A/TUR/2006 v/s). The nt variation was 5.0–7.0% when compared to the A/IRN/78/2009 v/s capsid sequence. The capsid amino acid sequences were also analysed and the aa variation was much higher compared to A22/Iraq (6.4–7.1%) than to A/TUR/2006 (3.0–4.1%) and A/IRN/78/2009 (3.0–4.4%).

Comparison between A22/Iraq, A/TUR/2006 and A/IRN/78/2009 v/s, and SIS-10 and -12 field isolates

In vitro testing of viruses belonging to the SIS-10 and SIS-12 sub-lineages with either A22/Iraq, A/TUR/2006 or A/IRN/78/2009 antisera generated low r1-values indicating lower expectancy of protection. The capsid aa sequences of these viruses along with two SIS-10 viruses reported in our previous study (see above) were analysed further to understand the molecular basis of the antigenic drift. The aa sequence of VP4 was found to be completely conserved except a single aa change in one virus (isolated in 2010) whereas multiple changes were observed in the other three viral proteins. As most of these SIS-10 and SIS-12 viruses exhibited poor cross-reactivity with the three BVS used in this study, we specifically looked for amino acid residues in the capsid sequence of the field isolates which were different from those of the vaccine strains. A total of three aa residues (VP1-96, VP2-191 and VP3-65) were identified; one each in the three surface-exposed capsid proteins, VP1-3 (Fig. 2B). All these residues are surface exposed (Fig. 2D) and could explain the inability of the antisera to cross-react with the field isolates, and are probably the sites prone to change to help the virus escape immune pressure. Out of these, VP2-191 has been reported to be part of a neutralising epitope (antigenic site-2) both in serotype O and A FMDV [9], [10]. Further, epitopes involving antigenic site 2 have been reported to be dominant within polyclonal responses of vaccinated animals [11]. Similarly, VP3-65 was identified in our previous study in viruses belonging to the BAR-08 sub-lineage [3]. Further, several residues in VP3 58–71 (antigenic site-4) have been reported to contribute to an epitope in serotype A either by monoclonal antibody (mAb) neutralisation resistant (mar) mutant studies [12] or by using structure-based epitope prediction softwares [13]. The change in VP1-96 has not been associated with antigenic significance, however, it is located in close proximity to the C-terminus of VP1 (of an adjacent protomer) that constitutes antigenic site 1 (Fig. 2E), and could be part of previously identified epitopes.

In addition, five capsid amino acid substitutions, two in VP3 (positions 70 and 175) and three in VP1 (positions 83, 85 and 171), were observed in ten out of 15 viruses (boxed grey in Fig. 2B) which may have some impact on the antigenic nature of the virus. Interestingly, these changes at five different positions were only observed in SIS-10 viruses. Out of these, VP1-83 was identified as a likely contributor to antigenic change in our previous study of viruses belonging to the BAR-08 sub-lineage [3] and was reported previously as an epitope in A12 virus by mar mutant studies [14]. Though VP1-171 has never been reported to be of antigenic significance, residues 169 and 173 located close to residue 171 were reported to be epitopes in A10 and A12 viruses by mar mutant studies, respectively [12], [14]. The two residues in VP3, positions 70 and 175 were reported as epitopes in serotype A viruses, A10 and A12 viruses, respectively by mar mutant studies [12], [14]. Thus, it appears that the residues within or close to neutralising antigenic sites are first targeted by the immune pressure in FMDV in an attempt to adapt to the changing environment.

Our previous study [3] identified eight aa residues as responsible for the antigenic drift of the A-Iran-05 viruses from A/TUR/2006 v/s, out of which four (VP1-28, 141; VP2-134 and VP3-220) were also implicated in this study (residues in bold and positions underlined in Fig. 2B–D). Of these, VP2-134 has been reported to influence binding of antigenic site 2 mAbs in serotype O viruses [15] and VP1-141 has been reported to be critical in serotype A mar mutant studies involving antigenic site 1 [16].

Serological cross-reaction between recent field isolates and putative vaccine strain

The poor cross-reactivity of the recent field viruses isolated in 2012 and 2013 with two existing vaccine strains and one putative vaccine strain strongly suggests that these v/s are not suitable for use in the region for FMD control. Therefore, development of new vaccine strains should be a priority to help control the serotype A outbreaks in this region. As a first step, we selected a more recent isolate from SIS-10 sub-lineage, and raised antisera in cattle. The pooled antisera (from 5 cattle) were used in serological assays to assess their suitability for development as a new v/s. All the viruses employed in this study exhibited a good match with the new antisera (Fig. 3) indicating its potential as a candidate v/s, subject to fulfilment of other criteria, such as suitable production and stability characteristics, and in vivoevaluation in the target host.

Fig. 3

Antigenic relationship (r1) values of SIS-10 and SIS-12 viruses with A/IRN/07/2013 strain. The horizontal dotted line indicates the cut-off value of 0.3, above which the vaccine is considered to be a good match.

In summary, analysis of the genetic and antigenic data of the SIS-10 and -12 viruses revealed substitutions in antigenic sites, especially site 1, 2 and 4 that either individually or together could underpin the antigenic drift of these type A viruses. Mutagenesis studies involving a cDNA clone could help to confirm these observations. The high rate of evolution in FMDV results in the emergence of new sub-lineages of serotype A viruses in the Middle East, requiring development of a new v/s every 5–10 years. Therefore, close monitoring of the outbreak strains in this area is essential to enable appropriate vaccines to be selected, and the need to develop a new v/s should be identified in a timely fashion to prevent future outbreaks. The results from this study highlight the continuing need for comprehensive surveillance and the urgency for development of a new serotype A vaccine for use in control programmes in this region.