Molecular epidemiology of foot-and-mouth disease (review)

INTRODUCTION

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, that may cause epizootics and inflict dramatic economic damage.

The causative agent of the disease is a non-enveloped RNA virus, which belongs to Aphthovirus genus of the Picornaviridae family. There are seven viral serotypes: O, A, C, Asia-1, SAT-1, SAT-2 and SAT-3.

Currently, the disease is widespread in Africa, Asia and some parts of South America, in which it causes huge economic losses to the agricultural sector. For the FMD-free countries there is still a danger of the disease introduction from the infected regions.

FMD is characterized by a very dynamic and complex epidemiology. The FMD virus (FMDV) is rapidly evolving, new genetic and antigenic variants of the virus are constantly emerging that can overcome the immunity of vaccinated animals and cause outbreaks of the disease. This significantly complicates the control of the disease and determines the need to constantly monitor the FMDV diversity in nature and to timely develop new vaccines adapted to emerging viral lineages.

Global FMD surveillance is performed by the FMD reference laboratory network of the World Organization for Animal Health and the Food and Agriculture Organization of the United Nations (WOAH/FAO) [1], with the main task to monitor the emergence of new FMDV variants with modified genetic and antigenic properties which render the available vaccines ineffective. An important surveillance tool is the molecular genetic analysis of the FMDV.

THE HISTORY OF THE DEVELOPMENT OF MOLECULAR EPIDEMIOLOGICAL TECHNIQUES

Molecular epidemiology as a scientific field is the combination of molecular biology, epidemiology and population genetics that has become a powerful tool to develop control strategies for infectious diseases [2].

Historically, the first attempts to use laboratory methods in epidemiological studies of FMD were associated with serological tests. More recently, biochemical methods such as FMDV SP polyacrylamide gel electrophoresis and T1 oligonucleotide fingerprinting of the viral genome began to be used [3-6]. However, the resolution of these methods was low, and they were not widely used.

A revolutionary event in the molecular epidemiology of foot-and-mouth disease was the use of sequencing technologies and phylogenetic analysis of the viral genome sequences. In 1987 E. Beck and K. Strohmaier showed that sequencing and comparative analysis of the VP1 gene of FMDV field isolates from the outbreak reveal their origin [7]. In particular, they established that 14 out of 18 FMD outbreaks in Western European countries between 1964 and 1985 were caused by vaccine strains. In most cases they were induced by improperly inactivated FMD vaccines. Several more outbreaks could occur as a result of the virus escape from the vaccine production plants. The German scientists suggested that nucleotide sequence analysis should be used as a standard method of diagnosis, because when compared with other techniques it more clearly reveals the origin and course of epizootics and offers the possibility of preventing further outbreaks. Beck E. and Strohmaier K. also proposed to prohibit the use of formalin-inactivated FMD vaccines, since they are the main source of FMDV in Europe. After this proposal was implemented, no FMD cases have been reported in Europe.

Since the late 1980s and early 1990s, nucleotide sequence analysis has been widely used to characterize FMDV field isolates [8–28].

Initially, the so-called direct RNA sequencing method was used for these purposes [29]. It included such preparatory steps as FMDV propagation in cell culture, purification and concentration of the virus and viral RNA, and therefore the method was very laborious and time-consuming. In addition, it allowed the determination of only 130–150 nucleotide bases of the FMDV VP1 gene. Since the mid-1990s, polymerase chain reaction (PCR) has been used to amplify genetic material for sequencing [30][31]. The PCR use has literally revolutionized the FMDV nucleotide analysis technology: the time needed for testing was reduced from 1–2 weeks to 1–2 days, it became possible to produce the full genome sequence of the VP1 gene and even of the FMDV. To sequence the FMDV full genome, E. M. Cottam et al. amplified 24 overlapping viral genome fragments using PCR [32]. Further progress in full genome sequencing was associated with the use of NGS (next-generation sequencing) technology [33].

STUDY OF THE FMDV GENETIC DIVERSITY

An impressive bank of nucleotide sequences of FMDV field isolate VP1 genes from different countries has been created at the World Reference Laboratory for Foot-and-Mouth Disease (Pirbright, UK) and the WOAH/FAO FMD regional reference laboratory network for FMD. The analysis of this database made it possible to evaluate the virus genetic diversity, to determine the viral evolution patterns in the endemic regions, and track the pathways of the FMD epizootic spread.

Molecular biological studies have shown that there are several levels of the FMDV genetic diversity: types, topotypes, genetic lineages and sublineages. The division of the virus into types fully corresponds to its classification into serotypes, that is, at the genetic level, as well as at the antigenic level, seven groups of the virus are distinguished, defined as types: A, O, C, Asia-1, SAT-1, SAT-2 and SAT-3. VP1 varies by 30–50% between the types [34].

Within each serotype, A. R. Samuel and N. J. Knowles [35] identified geographical types, which they called topotypes. VP1 nucleotide differences between the topotypes are up to 15–20%. It is obvious that the topotypes were formed as a result of the relatively independent FMDV evolution in certain geographical regions. The figure shows current FMDV pools. The FMDV division into topotypes generally corresponds to the division of the viral distribution into pools [36].

There are 11 topotypes within type O lineage: Cathay (China), SEA (Southeast Asia), ME-SA (Middle East and South Asia), Euro-SA (Europe and South America), WA (West Africa), EA-1 (East Africa-1), EA-2 (East Africa-2), EA-3 (East Africa-3), EA-4 (East Africa-4) and two extinct Indonesian topotypes ISA-1 and ISA-2 [37-39].

FMD type A virus is differentiated into 3 topotypes: Euro-SA (Europe and South America), Asia and Africa. Within each of these three topotypes, a very large genetic diversity of the virus has been revealed.

The emergence of the topotypes within serotypes A and O common for Europe and South America is explained by the fact that FMD of these two types was introduced to the American continent from Europe in the 19^th century.

Antigenically FMD Asia-1 viruses are less diverse than other serotypes. All known Asia-1 isolates form the only topotype – Asia. Knowles N. J. and Samuel A. R. explain the relatively low genetic diversity of the the serotype by its more recent origin than the others, or a severe ‘bottleneck’ purification with only a single topotype surviving [34].

FMDV serotype C has not been detected since 2004 and is currently considered to be extinct. FMDV type C, like serotypes A and O, is presumed to have been introduced into South America from Europe. However, the results of molecular biological studies allowed the British scientists from the Pirbright Institute to assume that the serotype C was introduced to South America and evolved from type A in around 1870 [40]. Later, this serotype was introduced to Europe from South America, and from Europe it spread to Africa and Asia. The scientists assume that exactly the occurrence of serotype C viruses in South America is the reason for their lower suitability to the ecological conditions of the other continents, which prevented them from persistence in the Old World.

SAT-1, SAT-2 and SAT-3 viruses are clustered into 13 (from I to XIII), 14 (from I to XIV) and 5 (from I to V) topotypes, respectively [37-39].

The discovery of the viral topotypes and their distribution pattern formed the basis for global FMD control strategy, which suggests clustering of seven regional alliances (seven regional FMDV pools) of FMD infected countries and coordination of national roadmaps for FMD progressive control pathway [41].

The FMDV is continuously evolving within each topotype, generating new genetic lineages that force out previously existing ones. Some lineages extinct quite quickly after their emergence, while others are able to circulate in their pools for a long time. The viral divergence within such “long-lived” genetic lineages leads to the emergence of sublineages. For example, by 2021, 17 sublineages were distinguished within the genetic lineage A/Iran-05, and 8 sublineages within O/PanAsia-2 [42].

It was found that FMDV genetic lineages evolve at very similar rates, estimated at an average of 1.3 × 10⁻² nucleotide substitutions/site/year (min-max range of 1.1 × 10⁻² – 1.4 × 10⁻²). These results indicate that FMDV evolves under a strict molecular clock that is largely constant among the different serotypes [42].

STUDY OF FMD PANZOOTIC SPREAD

Usually, FMDV of a certain topotype and genetic lineage evolves within a certain area (pool), periodically causing regional epizootics. However, over the past 30 years, two FMD panzootics have occurred, invloving several continents.

Molecular epidemiological studies made it possible to trace in detail the movements of the most devastating FMD panzootic on record caused by O/ME-SA/PanAsia virus. The virus was first isolated from FMD outbreaks in northern India in 1990. From 1991 to 1997 the new lineage appeared to spread to other parts of India and in 1998 it spread to Bahrain, Iran, Jordan, Kuwait, Lebanon, Syria, Saudi Arabia, and Yemen. In 1999, the spread of the PanAsian virus in the Middle East continued (outbreaks in Israel, Turkey and the United Arab Emirates), and it started rapidly spreading eastward from Hindustan: in May 1999 China reported FMD outbreaks caused by this virus, and in June O/ME-SA/PanAsia virus was isolated in Taiwan. Towards the end of 1999, the PanAsia virus moved to Myanmar, Thailand, Vietnam, Laos and by April 2000 all mainland Southeast Asian countries had experienced outbreaks due to the new strain. In March 2000 FMDV type O appeared in South Korea and Japan, and in April in Mongolia and Primorsky Krai of the Russian Federation. It should be noted that these East Asian territories previously had been free from FMD for many decades [34][43][44].

FMD panzootic caused by O/ME-SA/PanAsia virus was not limited to Asia. In September 2000 FMD type O was first reported in the Republic of South Africa. The outbreak occurred near the port of Durban. The origin was traced to feeding pigs with uncooked swill from a ship arrived from Asia. In February 2001, PanAsia virus was introduced and caused devastation in the United Kingdom. To eradicate the outbreak 6.5 million animals were destroyed, and direct and indirect losses to the country amounted to about 8 billion pounds. From the UK FMD moved to Ireland, France and the Netherlands [45].

Another FMD epidemic, but less serious, started in 2013. It was caused by O/ME-SA/Ind-2001 genetic lineage and covered the entire Asian continent and North Africa. This virus was first isolated in India in 2001 [45]. By 2009 it had spread widely in the territory of Hindustan and diversified into five sublineages: a, b, c, d, and e. In 2009, the O/ME-SA/Ind-2001 virus moved beyond its original range for the first time and caused an outbreak in Iran. Since 2013, the virus became panzootic and caused extensive outbreaks in North Africa and the Middle East in 2013–2014 [46, 47, 48], in Southeast Asia in 2015 [49] and East Asia in 2016–2017 [50].

Using the nucleotide sequences of VP1 (n = 424) full genome (n = 74) of O/ME-SA/Ind-2001 isolates from different countries, K. Bachanek-Bankowska et al. [50] used phylodynamic and phylogeographic reconstructions to track the global spread of this virus. In particular, it was shown that FMD outbreaks in the Middle East and North Africa in 2013–2015 resulted from five independent introductions of O/Ind-2001d sublineage virus from Hindustan, and outbreaks in Saudi Arabia in 2016 were caused by two independent introductions of another sublineage – O/Ind-2001e.

In 2015, O/ME-SA/Ind-2001 virus started its spreading eastward from Hindustan: from Bangladesh to Vietnam, and then sublineage d was introduced to Laos, and sublineage e was introduced through Myanmar in 2016 to Thailand and Vietnam. After that O/Ind-2001e sublineage virus caused an FMD epizootic in East Asia, involving China, South Korea, Mongolia and the Zabaikalsky Krai of the Russian Federation.

In December 2021, O/ME-SA/Ind-2001 virus caused an outbreak in the Orenburg Oblast of the Russian Federation, and in January 2022 in the Republic of Kazakhstan [51].

This means that the panzootic caused by O/ME-SA/Ind-2001 virus had much in common with the previous one caused by O/ME-SA/PanAsia virus: both genetic lineages originated from the Indian subcontinent (Bangladesh, Bhutan, India and Nepal), in both cases the disease spread first to the west (to Central and Western Asia), and only then the virus was introduced into Southeast and East Asia. Both viruses demonstrated the ability to spread rapidly in regions where other type O genetic lines were enzootic, as well as to spread to normally FMD-free countries [50]. The emergence of FMD panzootics is probably a consequence of the economic globalization.

REGIONAL FMD EPIZOOTICS IN POOLS 1 AND 3

FMD panzootics, covering the entire Asia and even several continents, are a rather rare event. Most FMD epizootics never expand beyond their regional pools. Regional epizootics occur when antigenically modified FMDV variants appear that break through the existing population immunity [52].

The FMD situation in the Russian Federation is influenced by epizootic in Pools 1 (East and Southeast Asia) and 3 (West and Central Asia).

In Western and Central Asia (Pool 3), the largest regional epizootics were caused by A/Iran-05, O/PanAsia-2 and Asia1/Sindh-08 lineages.

The A/Iran-05 virus was first identified in Iran in 2003. In 2005 it spread widely in this country, and then caused an epizootic that covered Turkey, Afghanistan, Pakistan, Bahrain, Saudi Arabia, Jordan, and Iraq. Sporadic FMD outbreaks caused by this virus were reported in Israel, Lebanon, and Kuwait. A/Iran-05 has forced previously circulating A/Iran-96 and A/Iran-99 lineages out from the Middle East and is dominating in this region to the present. During this period, the virus diverged into many sublineages. Some sublineages (for example, Iran-05^BAR-08) became widespread, while others (for example, Iran-05^ARD-07 and Iran-05^EZM-07) were reported only in one country (Turkey). Iran-05^BAR-08 sublineage virus was introduced to Libya in 2009, and to Egypt in 2010 [53-57].

O/ME-SA/PanAsia-2 virus is derived from the O/ME-SA/PanAsia lineage, which became widespread in Western and Central Asia in the late 1990s. As an independent lineage, O/ME-SA/PanAsia-2 was first reported in Iran in 2006. In 2007, the virus of this lineage was introduced to Turkey, Afghanistan, Pakistan, Saudi Arabia, Israel, and then to other countries in the Middle East. In 2011, the virus from Turkey was introduced to Bulgaria. O/ME-SA/PanAsia-2 virus has been dominating in Pool 3 since 2007. During this time, it has diverged into several sublineages, among them sublineage PanAsia-2^SIS-10 is the most widespread [55-57]. In 2010, the virus of this sublineage spread beyond Western Asia and caused FMD outbreaks in Libya and Bulgaria [56].

Another significant regional FMD epizootic in Western and Central Asia was caused by the Asia1/Sindh-08 virus. It was first identified in 2008 in Pakistan, from where it spread to Afghanistan, Iran, Iraq, Bahrain and Turkey. The Asia1/Sindh-08 virus has forced three other Asia-1 genetic lineages out from the pool and is dominating this region to the present [55-57].

In 2021, A. Di Nardo et al. [42] reconstructed the evolutionary history and spatial dynamics of FMD in Western and Central Asia (Pool 3) over the last 20 years. Having analyzed the history of A/Iran-05, O/PanAsia-2 and Asia1/Sindh-08 genetic lineages, they showed they highlighted the pivotal role played by virus circulation in Pakistan, Iran, and Afghanistan. These countries represent primary conveyors of FMDV infection across the region and are important sites for generating genomic diversity in Western and Central Asia. Phylogeographic reconstructions further revealed three main patterns of transboundary movements of viruses in Pool 3: 1) the continuous virus interchange between Pakistan, Afghanistan, and Iran, likely constituting interconnected large metapopulations; 2) the key role of Iran as hub of virus diffusion to the west; and 3) the unidirectional migration of viruses from Iran toward Turkey. This spatial pattern of FMDV spread in the region is common to all its serotypes.

In the 20^th century, the region of Western and Central Asia (Pool 3) had an enormous impact on the FMD situation in Russia, however, in the 21^st century the main source of FMD for the Russian Federation was Pool 1, which includes East and Southeast Asia. This is explained by the drastic change in the FMD situation in this region. The FMD situation in most of the East Asia countries remained relatively favourable during the 20^th century: Japan was free since 1908, Korea since 1934, the Far East of the Russian Federation since 1964, Mongolia since 1973 [43]. To a large extent, this favourable situation was achieved by the buffer role of China, which separated these countries from Southeast Asia, where FMD of types O and A is enzootic. In China itself, probably only the O/Cathay virus has historically been enzootic. This virus has a unique feature: it is capable of causing clinical disease only in pigs [58]. Probably, the absence in China of FMDV capable of infecting cattle ensured the disease freedom in Mongolia, Korea, Japan and the Russian Far East during the 20^th century [59].

Globalization and the PRC economic development have changed the situation. In the late 1990s, FMD O/ME-SA/PanAsia virus was introduced and widespread in China, which probably moved from China to South Korea, Japan, Mongolia and the Primorsky Krai of the Russian Federation in 2000.

Numerous Asia-1 FMD outbreaks were reported first in China, and then in Russia (Amur Oblast, Primorsky and Khabarovsk Krais, Chita Oblast), Mongolia and North Korea in 2005–2006. The virus responsible for this epizootic had only 1.11–1.74% VP1 nucleotide difference from viruses from India collected in 1980–1981 and had no close relatives among the isolates circulating at that time [60][61]. The most probable cause explaining the extremely close relationship of the isolates from 2005–2006 and 1980–1981 is the use of the Indian strain-based vaccine in China [61][62].

In 2009–2010 East Asia was almost simultaneously covered by two epizootics caused by FMD A/Asia/Sea-97 and O/SEA/Mea-98 viruses. Both genetic lineages are enzootic in Southeast Asian countries. In 2009, the geographic range of A/Asia/Sea-97 expanded and caused outbreaks in six provinces of China, and in January 2010 in South Korea [63]. In 2013, the virus moved from the PRC territory to the RF Zabaikalsky Krai and Amur Oblast and Mongolia [64]. The O/SEA/Mea-98 virus was responsible for a larger epizootic in East Asia in 2010: FMD outbreaks caused by this strain were reported in China, Korea, Japan, Mongolia and the Zabaikalsky Krai of the Russian Federation [63][65].

Since 2016, O/ME-SA/Ind-2001 virus has probably spread through China to Russia, Mongolia and South Korea [50].

This means, if 25 years ago only FMD O/Cathay virus was enzootic in China, currently four more genetic lineages are circulating in the country: O/ME-SA/PanAsia, O/SEA/Mea-98, O/ME-SA/Ind-2001 and A/Asia/Sea-97. This poses a high risk of FMD introduction from China to Russia.

MOLECULAR EPIDEMIOLOGICAL STUDIES OF FOOT-AND-MOUTH DISEASE IN THE RUSSIAN FEDERATION

In the Russian Federation, FMDV phylogenetic studies are conducted at the Federal Centre for Animal Health (Vladimir), which is the WOAH Regional Reference Laboratory for FMD for Eastern Europe, Transcaucasia and Central Asia and the FAO Reference Center for FMD. Since 2005 the Federal Centre for Animal Health has been involved into the network of WOAH/FAO reference laboratories for FMD responsible for the global FMD surveillance, with the main task to monitor the emergence of new FMDV variants with modified genetic and antigenic properties which render the available vaccines ineffective. The identification of FMDV genetic lineages and variants with modified antigenic properties makes it possible to promptly develop new vaccines against them. Within its international responsibilities the Federal Centre for Animal Health analyzes molecular genetic and antigenic properties of FMDV isolates responsible for the FMD outbreaks in the Russian Federation, Central Asia and Transcaucasia. FMDV isolates that caused outbreaks in Russia, CIS countries and in Mongolia were characterized by molecular genetic methods at the Federal Centre for Animal Health in the period from 1995 to 2022 [51][59][62][64-68].

Studies showed that FMD outbreaks in post-Soviet Russia were caused by the introduction of the virus belonging to various serotypes, topotypes and genetic lineages from infected Asian countries (Table 1). For example, FMD O/Cathay, O/ME-SA/PanAsia, O/SEA/Mya-98, O/ME-SA/Ind-2001, Asia1, A/Asia/Sea-97 viruses were introduced from China to various regions of the Russian Federation. FMD outbreaks in the North Caucasus in 2013 were caused by the introduction of A/Asia/Iran-05 virus from Transcaucasia, and the introduction of O/ME-SA virus of a rare non-classified group from Central Asia caused outbreaks in the Republic of Bashkortostan in 2017. In 2021 O/ME-SA/Ind-2001 virus was probably introduced to the territory of the Russian Federation from Kazakhstan. Thus, the results of the studies conducted by the Federal Centre for Animal Health suggest a large genetic diversity of FMDV introduced to the Russian Federation.

Phylogenetic analysis of the virus isolates responsible for FMD outbreaks in Transcaucasia (Armenia and Georgia) and Central Asia (Kazakhstan, Kyrgyzstan, Tajikistan, Uzbekistan) revealed that they belong to genetic lineages that dominated Pool 3 at different periods: O/ME-SA/Iran-01, A/Asia/Iran-96, O/ME-SA/PanAsia, Asia1/VI, Asia1/II, A/Asia/Iran-05, O/ME-SA/PanAsia-2, Asia1/Sindh-08, A/Asia/G-VII (Table 2). In 2011–2013, FMD outbreaks caused by O/ME-SA/PanAsia and A/Asia/Sea-97 viruses, introduced from China were reported in eastern regions of Kazakhstan. In 2022, O/ME-SA/Ind-2001 virus was introduced from China to Kazakhstan.

FMD outbreaks in Mongolia were caused by the virus responsible FMD epizootics in Pool 1: O/ME-SA/PanAsia, O/SEA/Mya-98, A/Asia/Sea-97, O/ME-SA/Ind-2001.

Within the network of the WOAH/FAO reference laboratories for FMD, the Federal Centre for Animal Health shares information on FMDV field isolates responsible for oubreaks in various regions of the world. The VP1 nucleotide sequences and full genomes of FMDV isolates responsible for the outbreaks in Russia, CIS countries and Mongolia produced by the Federal Centre for Animal Health were used to track the spread of global and regional epizootics caused by O/ME-SA/PanAsia, Asia1/II, Asia1/V, O/SEA/Mya-98, A/Asia/Sea-97, O/ME-SA/Ind-2001 viruses [42][43][47][50][60][61].

The results of molecular epidemiological studies performed by the Federal Centre for Animal Health are taken into account for the strain selection to manufacture vaccines and vaccinate livestock in FMD risk areas.

CONCLUSION

Thirty-five years of experience in molecular epidemiology of foot-and-mouth disease proves that phylogenetic analysis of the viral genome nucleotide sequences is an effective tool to monitor FMD both on the regional and global levels. FMD surveillance, which makes it possible to track the emergence of new viral variants with modified genetic and antigenic properties, allows for the rapid development of new vaccines and thus for a significant improvement of FMD control performance.

The use of molecular biological methods has made it possible to progress significantly in understanding of FMD epidemiology, which is important for the development of effective control strategies. The discovery of FMDV topotypes and their distribution pattern formed the basis for global FMD control strategy, which suggests clustering of seven regional alliances (seven regional FMD pools) of infected countries and coordination of national roadmaps for FMD progressive control pathway.