Insights into the Molecular Epidemiology of Foot-and-Mouth Disease Virus in Russia, Kazakhstan, and Mongolia in Terms of O/ME-SA/Ind-2001e Sublineage Expansion

Foot-and-mouth disease (FMD) has long been recognized as a highly contagious, transboundary disease of livestock incurring substantial losses and burdens to animal production and trade across Africa, the Middle East, and Asia. Due to the recent emergence of the O/ME-SA/Ind-2001 lineage globally contributing to the expansion of FMD, molecular epidemiological investigations help in tracing the evolution of foot-and-mouth disease virus (FMDV) across endemic and newly affected regions. In this work, our phylogenetic analysis reveals that the recent FMDV incursions in Russia, Mongolia, and Kazakhstan in 2021–2022 were due to the virus belonging to the O/ME-SA/Ind-2001e sublineage, belonging to the cluster from Cambodian FMDV isolates. The studied isolates varied by 1.0–4.0% at the VP1 nucleotide level. Vaccine matching tests indicated that the vaccination policy in the subregion should be tailored according to the peculiarities of the ongoing epidemiologic situation. The current vaccination should change from such vaccine strains as O1 Manisa (ME–SA), O no 2102/Zabaikalsky/2010 (O/ME-SA/Mya-98) (r1 = 0.05–0.28) to strains that most closely antigenically match the dominant lineage O No. 2212/Primorsky/2014 (O O/ME-SA//Mya-98) and O No. 2311/Zabaikalsky/2016 (O ME-SA/Ind-2001) (r1 = 0.66–1.0).


Introduction
Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting domestic and wild cloven-hoofed animals manifesting with the development of vesicles, chiefly in the mouth and on the feet coupled with a fever [1]. The causative agent belongs to the Picornaviridae family and the Aphthovirus genus [2]. The viral genome is a single-stranded positive-sense RNA of approximately 8 kb in length, containing a single open reading frame encoding four structural proteins and eight nonstructural proteins (NSPs). The viral capsid comprises structural proteins VP1, VP2, VP3, and VP4, while the other NSPs are mainly involved in viral replication and pathogenesis [3]. The VP1 protein acts as the main immunogenic component of the FMDV virion [4].
FMD causes limited mortality and severe morbidity levels in infected herds, disrupting regional and international trade in animals and animal products [5,6]. The World Organization for Animal Health has included FMDV on its list of notifiable diseases due to severe impacts and significant production losses by FMDV [7]. Over 70% of the world's cattle population is affected by FMD, and it has attained endemic status in Africa, the Middle East, Asia, and limited areas of South America.
FMDV exists as seven immunologically distinct serotypes based on VP1: A, O, Asia-1, SAT-1, SAT-2, SAT-3, C with a diversity of topotypes, genetic lineages, and strains [8]. Within endemic regions, FMDV serotypes are not evenly distributed, with serotype O having the widest distribution [1]. There is no cross-protection between the different serotypes [9,10]; thus, infection or vaccination with one FMDV serotype does not protect against others [11].
The phylogenetic analysis of the VP1 genomic region identified 10 topotypes of the FMDV O serotype that occur in East Africa (EA 1-4), West Africa (WA), Europe-South America (EURO-SA), Indonesia (ISA 1-2), Middle East-South Asia (ME-SA), and Southeast Asia (SEA) [12]. In Southeast Asia, there are three cocirculating topotypes: the O/SEA/Mya-98 lineage, the CATHAY topotype, and the Middle East-South Asia (ME-SA) topotype [13][14][15]. However, a novel lineage, O/ME-SA/Ind-2001, appeared in India in 2001 [16] and has recently raised concerns due to its range expansions coupled with ongoing multiple transregional movements. Since 2001, it spread to Saudi Arabia and the United Arab Emirates in 2013 [17], Bahrain in 2015 [18], and North African countries, including Libya in 2013 [19], Tunisia and Algeria in 2014 [17], and Morocco in 2015 [20], Southeast and East Asia [21,22], and Cambodia and Pakistan in 2018 [23,24]. The alarming rate of expansion may lead to high risks of causing a pandemic and further spillover into regions up north.
The Russian Federation and the Republic of Kazakhstan are nonendemic nations, with FMD-free zones recognized by the OIE. In previous years, sporadic FMD cases were reported in Russia that were attributed to spillovers from infected domestic and wild clovenhoofed animals of neighboring countries. The isolates were found in 2014, 2016, 2018, 2019, and 2020 in the region of Zabaikalsky Krai of Russia, which shares its borders with China and Mongolia, and belonged to the O serotype, prevalent in China and Mongolia, and sporadic cases of the A serotype were detected in 2013 [25].
Interestingly, sporadic cases in Kazakhstan in 2007 and 2010 were caused by O/ME-SA/PanAsia2 isolates that had been transmitted from Central Asia. However, from 2011 to 2013, genetic lineages of O/ME-SA/PanAsia and A/Asia/Sea-97 circulating in China and SEA escaped and extended their range into Kazakhstan [26,27]. Following the emergence of O/ME-SA/PanAsia strains that derived from South Asia, this lineage rapidly spread and attained panzootic status [28].
In 2021-2022, FMD outbreaks were reported in Mongolia, Kazakhstan, and Russia (collectively referred to as subregions), which share borders with countries with an active circulation of O/ME-SA/Ind-2001 lineage strains, revealing the unprecedented transboundary and aggressive movement of FMD in a northward direction [29].
This work aims to describe and characterize the genetic and antigenic relationships of FMDV isolates from Russia, Kazakhstan, and Mongolia to gain insights into the molecular evolution of FMDV strains together with circulating strains from FMD-affected neighboring countries in the subregion.

Sampling and Control Measure Taken during the Outbreak
In this study, we analyzed samples from three countries with shared borders: the Russian Federation, Kazakhstan, and Mongolia.
In the Russian Federation, the FMD outbreak occurred in the village of Karagach, Belyaevskii rayon, Orenburg region in a backyard unit with 17 cattle and 22 sheep. A total of 2 cows were affected and exhibited clinical signs ( Figure 1A,B). In the Russian Federation, the FMD outbreak occurred in the village of Karagach, Belyaevskii rayon, Orenburg region in a backyard unit with 17 cattle and 22 sheep. A total of 2 cows were affected and exhibited clinical signs ( Figure 1A In December 2021, two biological samples (aphthous ulcers) were collected from FMD-infected cattle (from two cows owned by one owner) in the Orenburg oblast of the Russian Federation and submitted to the Federal Center for Animal Health (FGBI "AR-RIAH," Vladimir, Russia) for laboratory testing on 24 December 2021 ( Figure 2).  In December 2021, two biological samples (aphthous ulcers) were collected from FMDinfected cattle (from two cows owned by one owner) in the Orenburg oblast of the Russian Federation and submitted to the Federal Center for Animal Health (FGBI "ARRIAH," Vladimir, Russia) for laboratory testing on 24 December 2021 ( Figure 2). In the Russian Federation, the FMD outbreak occurred in the village of Karagach, Belyaevskii rayon, Orenburg region in a backyard unit with 17 cattle and 22 sheep. A total of 2 cows were affected and exhibited clinical signs ( Figure 1A In December 2021, two biological samples (aphthous ulcers) were collected from FMD-infected cattle (from two cows owned by one owner) in the Orenburg oblast of the Russian Federation and submitted to the Federal Center for Animal Health (FGBI "AR-RIAH," Vladimir, Russia) for laboratory testing on 24 December 2021 ( Figure 2).  The territory of the village of Karagach, Belyaevsky district, Orenburg region was previously included in the free zone without FMD vaccination without official recognition by the OIE. After studying the clinical lesions of the oral mucosa in the cattle, the probable timing of the animal disease was provisionally determined, namely, the period from 10 to 22 December 2021. To curb the outbreak, the following control measures were taken in Russia: disinfection, quarantine, the destruction of clinically ill animals, the quarantine and movement control of animals from around the outbreaks was imposed, surveillance in the threatened zone, ring emergency vaccination in response to the outbreak in the infected and risk zones, control of the viral reservoir in wild fauna, the official destruction of animal products, and the official disposal of carcasses and byproducts. To ease the implementation of control measures, the area was zoned into an outbreak zone (the location of the virus, transmission factors), an infected zone (the area within 5 km of infected animals), a risk zone (a zone within 30 km of the infected zone), and a surveillance zone (an area within 10 km of the risk zone).
In February 2022, three samples of an aphthous suspension were collected from FMDinfected cattle from outbreaks in the Mongolian aimags (regions) of Sukhbaatar, Khentii, and Khovd in September-October 2021 [30].
The following control measures were implemented in Mongolia: ring emergency vaccination in response to the outbreak in the infected and risk zones, the quarantine and control of animal movement within the country, disinfection, and surveillance outside the infected and/or protection zone.
Two clinical samples (aphthous suspension) from Kazakhstan (in Kiykta, in the Shetsky district of the Karaganda region) collected in January 2022 indicated a FMD outbreak upon detection of O/ME-SA/Ind-2001e sublineage [31]. FMD outbreaks in Orenburgskaya oblast (Russia) and Karagandinskaya oblast (Kazakhstan) attributable to O/ME-SA/Ind-2001, were documented in regions that have had a long history of freedom (no vaccination): since 1973 in Orenburgskaya oblast, and 2007 in Karagandinskaya oblast.
Before the outbreak of foot-and-mouth disease in 2022, the Karaganda region of the Republic of Kazakhstan was a part of a zone that was free of FMD without vaccination, and was officially recognized by the OIE in 2015. According to the characteristic lesions of the mucous membrane of the oral cavity of infected animals on the day of detection, and considering the incubation period of FMD, the dates of the disease were provisionally determined as 15-27 December 2021. Samples from the affected areas, saliva, and blood were collected from two sick animals belonging to one owner and suspected of having FMD, and sent to the National Veterinary Referral Center. After the FMDV genome had been detected using an inhouse reverse-transcription polymerase chain reaction, samples were sent for confirmation to the Regional OIE Reference Laboratory for FMD (FGBI ARRIAH).
Kazakhstan reported the following undertaken measures to control the disease: disinfection, quarantine, the destruction of clinically ill and susceptible incontact animals, ring emergency vaccination zoning in response to the outbreak in infected and risk zones, movement control, and surveillance outside the infected zone.
More information regarding the number of susceptible and infected cattle, and measures taken can be found at https://wahis.woah.org, accessed on 23 October 2022, according to the reports submitted by the veterinary services.
To assist in the molecular epidemiological analysis of FMD reported in this manuscript, The locations of the studied FMD outbreaks are shown in Figure 1.

RNA Extraction
RNA was extracted from a 10% suspension of ulcers using Trizol (invitrogen) following the manufacturer's instructions. Prior to RNA extraction, tissue samples were crushed with a sterile grinder to prepare a 10% suspension in PBS buffer.

PCR and Sequencing
The VP1 gene was amplified and sequenced using previously described primers [32]. Sequencing was performed using the BigDye Terminator Cycle Sequencing kit and the ABI Prism 3130 automatic sequencer (Thermo Fisher Scientific, USA). Full-length VP 1 nucleotide sequences were analyzed using the BioEdit software package. MEGA7 software with the maximum-likelihood estimation and neighbor-joining methods, and 1000 bootstrap replicas were used to construct dendrograms. Each sequencing reaction was carried out in five replicates to ensure the consistency of the resulting VP1 sequences.

Virus Isolation
FMDV was isolated and reproduced using continuous monolayer cell cultures of Siberian ibex kidney cells (PSGK-30) and pig kidney cells from Instituto Biologico-Rim Suino-2 (IB-RS-2). The infected cell cultures (CCs) were incubated at 37 • C until CPE had been evident. To adapt the virus, no more than three passages were performed in the CCs. A virus that gave 90-100% CPE within 18-24 h was considered adapted [33].
Viral infectivity titration using a micromethod in IB-RS-2 CC was performed in 96-well culture plates on a continuous IB-RS-2 cell culture (concentration of 0.8-1.0 × 10 6 cell/mL at 37 • C and 5% CO 2 for 48 h). The viral titer was read using an inverted microscope considering the number of wells with evident specific CPE, calculated with the Karber method, and expressed as log10 TCD 50 /50 µL (TCD, tissue cytopathic dose).

Vaccine Matching
Reference sera were obtained at the FGBI ARRIAH from cattle vaccinated with FMD monovalent inactivated vaccines from the following strains belonging to ME-SA topotypes at 21-30 days FMD viral isolates were matched with the production strain with the viral neutralization test (VNT) in IB-RS-2 CC. The titers of reference sera from cattle immunized with the vaccines on the basis of the production of homologous and heterologous FMDV strains were determined with the checkerboard titration method using five doses of the virus. The serum titer against 100 TCD 50 was estimated with regression and expressed as log10. An r 1 value was calculated as the reciprocal arithmetic log10 titer of reference serum against heterologous and homologous viruses, and was interpreted according to M. Rweyemamu (1984) [34]: r 1 ≥ 0.3 indicates a close antigenic relationship among the strains, so the use of a vaccine based on this production strain is likely to confer protection against challenges Viruses 2023, 15, 598 6 of 12 with the field isolate; r 1 < 0.3 indicates no antigenic relationship among the strains, and the production strain does not confer protection against the field isolate; r 1 = 0.28-0.32 was the cut-off value range. the vaccines on the basis of the production of homologous and heterologous FMDV strains were determined with the checkerboard titration method using five doses of the virus. The serum titer against 100 TCD50 was estimated with regression and expressed as log10. An r1 value was calculated as the reciprocal arithmetic log10 titer of reference serum against heterologous and homologous viruses, and was interpreted according to M. Rweyemamu (1984) [34]: r1 ≥ 0.3 indicates a close antigenic relationship among the strains, so the use of a vaccine based on this production strain is likely to confer protection against challenges with the field isolate; r1 < 0.3 indicates no antigenic relationship among the strains, and the production strain does not confer protection against the field isolate; r1 = 0.28-0.32 was the cut-off value range.

VP1 Sequencing
The FMD virus identified in the tested samples belonged to the ME-SA topotype (Middle East-South Asia), genetic lineage O/ME-SA/Ind-2001, according to VP1 sequenc-

VP1 Sequencing
The FMD virus identified in the tested samples belonged to the ME-SA topotype (Middle East-South Asia), genetic lineage O/ME-SA/Ind-2001, according to VP1 sequencing and phylogenetic analysis (Figure 3). At the VP1 gene, the isolates from Russia, Kazakhstan, and Mongolia differed by 1.0-3.8% (Table 1). The Mongolian isolates varied by 1.0-3.3%, whereas the Russian and Kazakhstan isolates varied by only 0.8%. The Cambodian isolates from 2019 (MZ634454, MZ634455, and MZ634456) displayed the closest genetic resemblance to the isolates analyzed from Russia, Mongolia, and Kazakhstan according to our analysis of the acquired VP1 sequences against the NCBI (Figure 4).

Vaccine Matching
The results of antigenic variation across the FMD viral isolates recovered in Russia, Mongolia, and Kazakhstan in 2021-2022 with a VNT assay are given in Table 2.

Vaccine Matching
The results of antigenic variation across the FMD viral isolates recovered in Russia, Mongolia, and Kazakhstan in 2021-2022 with a VNT assay are given in Table 2. The findings from Table 2

Discussion
This is the first study looking into genetic variation in FMDV circulating across a few countries in the Northern Hemisphere. The FMDV O/ME-SA/Ind-2001 lineage has pandemic potential, and now represents an important challenge to veterinary services and

Discussion
This is the first study looking into genetic variation in FMDV circulating across a few countries in the Northern Hemisphere. The FMDV O/ME-SA/Ind-2001 lineage has pandemic potential, and now represents an important challenge to veterinary services and farmers.
By 2021-2022, the FMDV O/ME-SA/Ind-2001 lineage rapidly spread over a long distance in a northward direction, and caused outbreaks in Kazakhstan, Mongolia, and Russia ( Figure 4). The sequence analysis carried out herein demonstrates a high level of genetic relatedness across the studied isolates that shared 1-4% differences, whereas isolates from Russia were closer to those from Kazakhstan than those from Mongolia (Table 1) Figure 4). Surprisingly, in December 2021, an FMD outbreak caused by the O/ME-SA/Ind-2001 virus was reported in the Orenburg oblast of the Russian Federation (Karagach village, Belyaevsky Raion) that, like all regions of Russia bordering Kazakhstan, had been free from FMD for several previous decades, and had not practiced vaccination for the previous two years (Figure 3). The genetic relationship (more than 99% VP1 similarity) of the FMDV O/Orenburg/2021/Rus and O/KAZ/2022 isolates, a short interval of time between the outbreaks, and the geographical location of the Russian FMD outbreak (19 km away from the state border with Kazakhstan) suggest a concurrent O/ME-SA/Ind-2001 FMDV spillover into both countries. This argues for a need to strengthen FMD surveillance using laboratory diagnostic methods in the population of wild migrating cloven-hoofed animals, which may be asymptomatic viral carriers that do not always manifest clinical signs when infected [36].
In Interestingly, antibodies to structural (Type O) and nonstructural FMDV proteins were detected in the population of Mongolian gazelles (Procapra gutturosa) migrating from Mongolia, pointing to the role of wildlife in transmitting FMDV [37].
The detection of the O/ME-SA/Ind-2001 genetic lineage virus in Kazakhstan, Russia, and Mongolia in 2021-2022 implies that the panzootic is escalating and expanding to new territories. The selection of a strain for the manufacture of vaccines effectively protecting animals is necessary in order to avoid the disease caused by the circulating the O/ME-SA/Ind-2001e sublineage, since this must be based on matching a representative field isolate from outbreaks in the region [37].
Several sociological and economic factors should be considered as contributing factors for the outbreaks of FMD. The Bayan-Ulgiy aimag is a territory of compact residence of Kazakhs on the territory of Mongolia, and its citizens constantly communicate closely with their relatives who moved to Kazakhstan after the break-up of the Soviet Union. According to the Mongolian database, 40,000 Mongolian citizens received Kazakhstani citizenship in the past 20 years (1995-2015). Therefore, the transmission of the infection via various routes is possible (transport, food, clothing, etc.).
In this study, we carried out a two-dimensional VNT utilizing reference monovalent bovine sera in order to compare the O/ME  Institutional Review Board Statement: Ethical review and approval were waived for this study due to the fact that no animals were handled in this study.