Genotyping of foot‐and‐mouth disease viruses collected in Sudan between 2009 and 2018

Abstract Foot‐and‐mouth disease (FMD) is widely distributed in Sudan where outbreaks occur on an annual basis especially during the winter months (December‐February). This study aimed to increase our understanding of the epidemiological patterns of FMD in Sudan and connections to neighbouring countries by characterizing the genetic sequences of FMD viruses (FMDV) collected from samples collected in 10 Sudanese states over a 10‐year period (between 2009 and 2018). FMDV was detected in 91 of the 265 samples using an antigen‐detection ELISA. Three serotypes were detected: O (46.2%), A (34.0%), and SAT 2 (19.8%). Fifty‐two of these samples were submitted for sequence analyses, generating sequences that were characterized as belonging to O/EA‐3 (n = 17), A/AFRICA/G‐IV (n = 23) and SAT 2/VII/Alx‐12 (n = 12) viral lineages. Phylogenetic analyses provided evidence that FMDV lineages were maintained within Sudan, and also highlighted epidemiological connections to FMD outbreaks reported in neighbouring countries in East and North Africa (such as Ethiopia and Egypt). This study motivates continued FMD surveillance in Sudan to monitor the circulating viral lineages and broader initiatives to improve our understanding of the epidemiological risks in the region.


INTRODUCTION
Foot-and-mouth disease (FMD) remains a worldwide major constraint to animal production and international trade. The causative agent, FMD virus (FMDV; genus Aphthovirus, family Picornaviridae), exists as seven immunologically distinct serotypes (O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3) and the virus is known to infect up to 70 cloven-hoofed animal species (Grubman & Baxt, 2004). The epidemiology of FMD in Africa is complex. This is partly because the continent is resident to three out of the seven endemic FMDV pools (Pools 4, 5 and 6;Paton et al., 2009), and six out of the seven FMDV serotypes have been recorded (Rweyemamu et al., 2008), although serotype C has not been detected since 2004 (Paton et al., 2021). Sudan is one of the largest African countries (nearly 1.9 million km 2 ) which has a resident population of more than 100 million FMD-susceptible animals. It has 30-40 million cattle, 70 million small ruminants as well as a diverse range of FMD susceptible wildlife species that are located mostly in the Southern areas or in the Dinder National Park in Eastern Sudan (Anon, 2009).
Sudan is recognized as an important crossroads between sub-Saharan and Northern Africa, and between East and West Africa.
FMD has been known to be present in Sudan since 1903(Abu Elzein, 1983) and previous studies have highlighted the contribution of FMD circulation in the country to the wider epidemiology of the disease in Africa (Bronsvoort et al., 2004;Hall et al., 2013;Rweyemamu et al., 2008;Ularamu et al., 2017). In sub-Saharan African countries, FMD is endemic but clinical signs are often mild or inapparent particularly in sheep and goats and outbreaks are inconsistently reported to veterinary services. Together with the costs and logistics associated with the shipment of suspected FMDV samples, these issues result in infrequent submissions to international reference centres [such as the World Reference Laboratory for FMD (WRLFMD), Pirbright, United Kingdom] and consequent potential for biases in epidemiological inferences (Bronsvoort et al., 2004). To address these sampling and reporting issues, the Government of Sudan together with the Food and Agriculture Organization of the United Nations (FAO) and the European Commission for the Control of FMD (EuFMD) joined efforts in a Technical Corporation Programme (TCP/SUD/3401) to promote active surveillance of FMD in Sudan which was undertaken between 2012 and 2014.
In this project extensive serosurveillance to detect antibodies against FMDV in cattle and small ruminants was carried out and the data has been reported elsewhere (Raouf et al., 2016(Raouf et al., , 2017

FMDV detection and serotyping
All samples were screened for the presence of FMDV antigen at the Central Veterinary Research Laboratory (CVRL) either using the indirect sandwich ELISA kit (WRLFMD; Roeder & Le Blanc Smith, 1987)  Unprocessed epithelium samples in the described transport medium were dispatched, under dry ice, as dangerous biological substance category B UN 3373. At the WRLFMD, samples were tested by real time RT-PCR (Callahan et al., 2002;Shaw et al., 2007) and virus isolation using primary bovine thyroid and IB-RS-2 cells as previously described (de Castro, 1964;Snowdon, 1966). Samples were considered FMDV negative if no CPE was observed for 48 h following blind passage of the first cell cultures after 48 h incubation at 37 • C. Samples in which FMDV was identified (n = 91) are listed in Table 1 together with associated metadata; those lacking a WRLFMD reference number (n = 38) were not submitted to the WRLFMD.
Sanger sequencing was performed on an ABI 3730xl DNA Analyzer (ABI Biosystems, Waltham, Massachusetts). Complete VP1 nucleotide sequences were aligned using BioEdit 7.0.5.3 (Hall, 1999) and Clustal W 1.83 (Thompson et al., 1994). Optimal nucleotide substitution models were computed for each serotype using MEGA 7 (Kumar et al., 2016). The maximum likelihood algorithm was used to construct phylogenetic trees using MEGA 7. One thousand bootstrap pseudoreplicates were used to assess branching confidence.

Phylogenetic analysis
All FMDV positive samples generated a single VP1 sequence apart from SUD/7/2017 where a mixture of O and SAT 2 were recovered.

Serotype O
All the eighteen serotype O viruses that were genotyped during this study fell within the O/EA-3 topotype ( Figure 2). These sequences represent at least four distinct clades (indicated by grey arrows in

Serotype A
Twenty-three serotype A viruses belonging to the A/AFRICA/G-IV lineage were genotyped ( Figure 3) during this study. Sudanese FMDVs, collected during 2018, belonged to two genetic clades (12.8-14.6% nt difference), with older ones represented in ancestral clusters. One of these contemporary clusters containing A/SUD/6-9/2018 also con-

Serotype SAT 2
Twelve serotype SAT 2 viruses within the SAT 2 topotype VII, distributed between 2010 and 2017 were characterized (Figure 4). All of these Sudanese viruses belonged to the Alx-12 lineage which also contains sequences from FMDVs collected from other countries includ-  et al., 2010a;Raouf et al., 2009). Serotype O was the most frequently detected; findings which are also supported by recent serological data (Raouf et al., 2016) showing higher prevalence of serotype O specific antibodies (60.2%) compared to serotype A (30.0%) and serotype SAT 2 (12.3%). Nucleotide distances of more than 15% (for serotypes O and A) or 20% (for serotypes SAT 1 and SAT 2) are used to classify isolates into different topotypes (Knowles & Samuel, 2003;Samuel & Knowles, 2001), while nucleotide differences of between 5% and 15% indicate distinct virus lineages (Bronsvoort et al., 2004). The Sudanese sequences reported here differed from the prototype strains of the relevant topotypes (Knowles et al., 2016) (Habiela et al., 2009) and Blue Nile State (Raouf, 2015;Raouf et al., 2017). This may indicate a varied role of sheep in the epidemiology of FMD. FMD infection in wild ruminants in Sudan has not been investigated.
In summary, FMD infection in Sudan remains regionally significant and this study highlights the epidemiological connections between