Outbreak investigations and molecular characterization of foot‐and‐mouth disease viruses circulating in south‐west Niger

In Niger, the epidemiological situation regarding foot‐and‐mouth disease is unclear as many outbreaks are unreported. This study aimed (i) to identify Foot‐and‐mouth disease virus (FMDV ) strains currently circulating in cattle herds, and (ii) to identify risk factors associated with Foot‐and‐mouth disease (FMD )‐seropositive animals in clinical outbreaks. Epithelial tissues (n  = 25) and sera (n  = 227) were collected from cattle in eight districts of the south‐western part of Niger. Testing of clinical material revealed the presence of FMDV serotype O that was characterized within the O/WEST AFRICA topotype. The antigenic relationship between one of the FMDV isolates from Niger (O/NGR /4/2015) and three reference vaccine strains was determined by the two‐dimensional virus neutralization test (2dmVNT ), revealing a close antigenic match between the field isolate from Niger and three FMDV serotype O vaccine strains. Serological analyses using a non‐structural protein (NSP ) test provided evidence for previous FMDV infection in 70% (158/227) of the sera tested. Multivariate logistic regression analysis revealed that only the herd composition (presence of both cattle and small ruminants) was significantly associated with FMDV seropositivity as defined by NSP ‐positive results (p ‐value = .006). Of these positive sera, subsequent testing by liquid‐phase blocking ELISA (LPBE ) showed that 86% (136/158) were positive for one (or more) of four FMDV serotypes (A, O, Southern African Territories (SAT ) 1 and SAT 2). This study provides epidemiological information about FMD in the south‐western part of Niger and highlights the complex transboundary nature of FMD in Africa. These findings may help to develop effective control and preventive strategies for FMD in Niger as well, as other countries in West Africa.     

Isolation,identification and retrospective study of foot‐and‐mouth disease virus from affected Mithun (Bos frontalis) in north‐eastern India

Foot‐and‐mouth disease (FMD ) is a contagious disease of cloven‐hoofed animals that causes substantial and perpetual economic loss. Apart from the contagious nature of the disease, the FMD virus can establish in a “carrier state” among all cloven‐hoofed animals. The Mithun (Bos frontalis ), popularly called the “Cattle of Mountain,” is found in the geographically isolated, hilly region of north‐east India: Arunachal Pradesh, Nagaland, Manipur and Mizoram. Despite the geographical inaccessibility, infection by FMD virus has emerged as the single most devastating disease among Mithun after the eradication of rinderpest from this region. Samples from outbreaks of FMD in Mithun were analysed by sandwich ELISA , multiplex RT ‐PCR (MRT ‐PCR ) and liquid‐phase blocking enzyme‐linked immunosorbent assay and isolated in the BHK ‐21 cell line. The results indicate the presence of FMDV serotype “O.” The sequencing and molecular phylogenies have revealed close relationships in the lineage of type “O” isolates from Bangladesh. The findings will provide useful information for further research and development of a sustainable programme for the progressive control of FMD in the Mithun population.     

A Refined Guinea Pig Model of Foot‐and‐Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds

An antiviral containment strategy for foot‐and‐mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre‐emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV‐infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T‐1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T‐1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID₅₀ of the GP‐adapted FMDV strain O₁ Manisa 1 h after the first administration. The efficacy of T‐1105 was compared with that of prophylactic vaccination with a highly potent double‐oil emulsion‐inactivated O₁ Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T‐1105‐treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T‐1105‐treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T‐1105‐treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T‐1105‐treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T‐1105 conferred a substantial clinical and virological protection against infection with O₁ Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.     

Early Detection of Foot‐And‐Mouth Disease Virus from Infected Cattle Using A Dry Filter Air Sampling System

Foot‐and‐mouth disease (FMD) is a highly contagious livestock disease of high economic impact. Early detection of FMD virus (FMDV) is fundamental for rapid outbreak control. Air sampling collection has been demonstrated as a useful technique for detection of FMDV RNA in infected animals, related to the aerogenous nature of the virus. In the current study, air from rooms housing individual (n = 17) or two groups (n = 4) of cattle experimentally infected with FDMV A24 Cruzeiro of different virulence levels was sampled to assess the feasibility of applying air sampling as a non‐invasive, screening tool to identify sources of FMDV infection. Detection of FMDV RNA in air was compared with first detection of clinical signs and FMDV RNA levels in serum and oral fluid. FMDV RNA was detected in room air samples 1–3 days prior (seven animals) or on the same day (four animals) as the appearance of clinical signs in 11 of 12 individually housed cattle. Only in one case clinical signs preceded detection in air samples by one day. Overall, viral RNA in oral fluid or serum preceded detection in air samples by 1–2 days. Six individually housed animals inoculated with attenuated strains did not show clinical signs, but virus was detected in air in one of these cases 3 days prior to first detection in oral fluid. In groups of four cattle housed together, air detection always preceded appearance of clinical signs by 1–2 days and coincided more often with viral shedding in oral fluid than virus in blood. These data confirm that air sampling is an effective non‐invasive screening method for detecting FMDV infection in confined to enclosed spaces (e.g. auction barns, milking parlours). This technology could be a useful tool as part of a surveillance strategy during FMD prevention, control or eradication efforts.     

Foot‐and‐mouth disease virus transmission dynamics and persistence in a herd of vaccinated dairy cattle in India

Foot‐and‐mouth disease (FMD ) is an important transboundary disease with substantial economic impacts. Although between‐herd transmission of the disease has been well studied, studies focusing on within‐herd transmission using farm‐level outbreak data are rare. The aim of this study was to estimate parameters associated with within‐herd transmission, host physiological factors and FMD virus (FMDV ) persistence using data collected from an outbreak that occurred at a large, organized dairy farm in India. Of 1,836 regularly vaccinated, adult dairy cattle, 222 had clinical signs of FMD over a 39‐day period. Assuming homogenous mixing, a frequency‐dependent compartmental model of disease transmission was built. The transmission coefficient and basic reproductive number were estimated to be between 16.2–18.4 and 67–88, respectively. Non‐pregnant animals were more likely to manifest clinical signs of FMD as compared to pregnant cattle. Based on oropharyngeal fluid (probang) sampling and FMDV ‐specific RT ‐PCR , four of 36 longitudinally sampled animals (14%) were persistently infected carriers 10.5 months post‐outbreak. There was no statistical difference between subclinical and clinically infected animals in the duration of the carrier state. However, prevalence of NSP ‐ELISA antibodies differed significantly between subclinical and clinically infected animals 12 months after the outbreak with 83% seroprevalence amongst clinically infected cattle compared to 69% of subclinical animals. This study further elucidates within‐herd FMD transmission dynamics during the acute‐phase and characterizes duration of FMDV persistence and seroprevalence of FMD under natural conditions in an endemic setting.     

Quantitative characteristics of the foot‐and‐mouth disease carrier state under natural conditions in India

The goal of this study was to characterize the properties and duration of the foot‐and‐mouth disease (FMD ) carrier state and associated serological responses subsequent to vaccination and naturally occurring infection at two farms in northern India. Despite previous vaccination of cattle in these herds, clinical signs of FMD occurred in October 2013 within a subset of animals at the farms containing juvenile‐yearling heifers and steers (Farm A) and adult dairy cattle (Farm B). Subsequent to the outbreak, FMD virus (FMDV ) asymptomatic carriers were identified in both herds by seroreactivity to FMDV non‐structural proteins and detection of FMDV genomic RNA in oropharyngeal fluid. Carriers’ seroreactivity and FMDV genome detection status were subsequently monitored monthly for 23 months. The mean extinction time of the carrier state was 13.1 ± 0.2 months, with extinction having occurred significantly faster amongst adult dairy cattle at Farm B compared to younger animals at Farm A. The rate of decrease in the proportion of carrier animals was calculated to be 0.07 per month. Seroprevalence against FMDV non‐structural proteins decreased over the course of the study period, but was found to increase transiently following repeated vaccinations. These data provide novel insights into viral and host factors associated with the FMDV carrier state under natural conditions. The findings reported herein may be relevant to field veterinarians and governmental regulatory entities engaged in FMD response and control measures.     

Direct detection and characterization of foot‐and‐mouth disease virus in East Africa using a field‐ready real‐time PCR platform

Effective control and monitoring of foot‐and‐mouth disease (FMD ) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE ). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD ‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR ™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT ‐PCR (rRT ‐PCR ) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT ] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE ‐recommended laboratory‐based rRT ‐PCR (determined using a panel of 57 FMDV ‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV ‐typing assay was able to correctly identify the serotype of 33/36 FMDV ‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP ) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n  = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT ‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.     

Challenges for Serology‐Based Characterization of Foot‐and‐Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011

Control of foot‐and‐mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK^® FMDV NS ELISA, solid‐phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK^® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti‐NSP positive cattle sera. However, 35% of the anti‐NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa‐2 (EA‐2) topotype, each differing from the currently used vaccine strain (EA‐1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT‐SPBEs, perform vaccine matching and implement improved regional FMD control.     

First detection of foot‐and‐mouth disease virus O/ME‐SA/ Ind2001e sublineage in Jordan

Foot‐and‐mouth disease (FMD) is a highly contagious vesicular disease that is caused by the FMD virus (FMDV). This disease affects both wild and domestic cloven‐hoofed animals, and the latter of which includes cattle, swine, sheep and goats. FMD is endemic to Jordan and has a severe impact on the productivity of domestic livestock. In January 2017, FMD outbreaks were detected in different animal species across Jordan, resulting in high mortality rates among young lamb and goat populations as well as causing classic FMD symptoms in cattle. In this study, clinical specimens were collected from animals affected by FMD. The results obtained from sequencing the VP1 gene place the studied FMDV isolate within the FMDV O/ME‐SA/ Ind2001e sublineage. Phylogenetic analysis of VP1 suggests that the O/JOR/1/2017 isolate is very similar to that of viruses isolated from Saudi Arabia in 2016. The possible introduction of this strain to Jordan might occur through transboundary animal movement or other transmission routes from Saudi Arabia, a neighbouring country.     

The Pathogenesis of Foot‐and‐Mouth Disease II: Viral Pathways in Swine,Small Ruminants,and Wildlife; Myotropism,Chronic Syndromes,and Molecular Virus–Host Interactions

Investigation into the pathogenesis of foot‐and‐mouth disease (FMD) has focused on the study of the disease in cattle with less emphasis on pigs, small ruminants and wildlife. ‘Atypical’ FMD‐associated syndromes such as myocarditis, reproductive losses and chronic heat intolerance have also received little attention. Yet, all of these manifestations of FMD are reflections of distinct pathogenesis events. For example, naturally occurring porcinophilic strains and unique virus–host combinations that result in high‐mortality outbreaks surely have their basis in molecular‐, cellular‐ and tissue‐level interactions between host and virus (i.e. pathogenesis). The goal of this review is to emphasize how the less commonly studied FMD syndromes and host species contribute to the overall understanding of pathogenesis and how extensive in vitro studies have contributed to our understanding of disease processes in live animals.     

Molecular Characterisation of Foot‐and‐Mouth Disease Viruses from Pakistan, 2005–2008

Foot‐and‐mouth disease (FMD), an economically important disease of cloven‐hoofed animals, is endemic in Pakistan where three virus serotypes are present (O, A and Asia 1). Fifty‐eight clinical samples collected between 2005 and 2008 from animals with suspected FMD in various locations in Pakistan were subjected to virus isolation on primary cell culture, antigen ELISA and real‐time RT‐PCR (rRT‐PCR). Viruses were isolated from 32 of these samples and identified as FMDV type O (n = 31) or type A (n = 1). Foot‐and‐mouth disease virus (FMDV) genome was detected in a further 11 samples by real‐time RT‐PCR. Phylogenetic analyses of the VP1 nucleotide sequences showed that all of the type O viruses belonged to the MIDDLE EAST–SOUTH ASIA topotype with the majority belonging to the PanAsia‐2 lineage; a single example of the older PanAsia lineage was identified. The single FMDV type A virus belonged to the ASIA topotype, but did not cluster with known strains that are currently circulating (such as Iran‐05) and was not closely related to other type A viruses from the region. These findings demonstrate the widespread distribution of O‐PanAsia‐2 in Pakistan and the presence of undisclosed novel type A lineages in the region.     

Herd Reproduction Ratio and Time–Space Analysis of a Foot‐and‐mouth Disease Epidemic in Peru in 2004

The herd reproductive ratio (R_h) and spatio‐temporal clustering were estimated in the 2004 foot‐and‐mouth disease (FMD) epidemic in Peru. The epidemic lasted 39 days and involved 26 herds. Movement of cattle was restricted, all susceptible species within a 25‐km buffer zone were revaccinated, and infected animals with clinical signs of FMD were killed or destroyed to control and eradicate the disease. The R_h declined from 5.3 on the second day of the epidemic to 1.31 on the 25th day. Spatio‐temporal clustering of cases was detected at a critical distance of 0.5 km and critical times of 7 and 14 days. Cases were clustered in space (P = 0.006) but not in time (P = 0.498). The space–time scan method detected a spatio‐temporal cluster that included consecutive case numbers 13, 14 and 15, located at the temporal midpoint of the epidemic. The values estimated for R_h and the cluster analyses provide quantitative estimates of the self‐limiting nature of FMD spread in a susceptible but vaccinated population.     

A Review of OIE Country Status Recovery Using Vaccinate‐to‐Live Versus Vaccinate‐to‐Die Foot‐and‐Mouth Disease Response Policies I: Benefits of Higher Potency Vaccines and Associated NSP DIVA Test Systems in Post‐Outbreak Surveillance

To rapidly return to trade, countries with OIE status, FMD‐free country where vaccination is not practised, have destroyed emergency vaccinated animals, raising ethical concerns with respect to social values, the environment, animal welfare and global food security. This two‐part review explores whether science could support eligibility to return to previous OIE status in 3 months irrespective of vaccinate‐to‐live or vaccinate‐to‐die policies. Here, we examine the benefits of higher potency (≥ 6 PD₅₀), high‐purity vaccines formulated from antigen banks for emergency use, their efficacy and performance in differentiating infected from vaccinated animals (DIVA) assays for post‐outbreak surveillance. From an intensive programme of research, we conclude that high‐quality, higher potency vaccines are proven to reduce FMD virus (FMDV) subclinical circulation and the risk of carriers. Broader coverage than predicted by serology suggests the potential to hold a few ‘key’ vaccine strains improving logistics and reducing the financial burden of antigen banks. The OIE should adopt formal definitions for emergency vaccination and emergency vaccines. In terms of supportive tools, we consider that the lack of OIE recognition of DIVA tests other than those of PANAFTOSA in cattle is a shortcoming. There is need for research on maternal antibody interference with DIVA tests and on the use of such tests to establish whether greater purification of vaccines improves performance. We consider that alignment of waiting periods for vaccinate‐to‐live and vaccinate‐to‐die in OIE Code Article 8.5.9 1 b. and c. is feasible until an acceptable level of statistical certainty for surveillance or target probability of freedom is established to substantiate the absence of FMDV infection or circulation. It is surveillance intensity rather than waiting periods that establishes the risk of residual FMDV. EU Directive 2003/85/EC implicitly recognizes this, permitting derogation of the OIE waiting periods.     

Foot‐and‐mouth disease outbreaks in captive scimitar‐horned oryx (Oryx dammah)

This paper describes three episodes of foot‐and‐mouth disease (FMD) that were detected during 2013–2015 in scimitar‐horned oryx (Oryx dammah) (SHO), a large Sahelo‐Saharan antelope extinct in the wild housed in a wild ungulate breeding facility located 50 km east of Abu Dhabi, United Arab Emirates. While no mortality attributable to FMD was noted in the population of nearly 4,000 SHO during two of the three outbreaks, the morbidity varied according to the circulating strains and seroconversion reached a plateau of 78.0% within two weeks and remained at this level for at least nine months. Partial or complete sequencing of the VP1 encoding region demonstrated that the three outbreaks were caused by three different FMDV lineages (O/ME‐SA/PanAsia‐2, A/ASIA/Iran‐05 and O/ME‐SA/Ind‐2001), consistent with FMD viruses that are circulating elsewhere in the region. These findings demonstrate that SHO are susceptible to FMD and highlight the risks of virus incursion into zoos and captive facilities in the Arabian Peninsula.     

Antibodies Against Foot‐and‐mouth Disease (FMD) Virus in African Buffalos (Syncerus caffer) in Selected National Parks in Uganda (2001–2003)

In East Africa, the foot‐and‐mouth disease (FMD) virus (FMDV) isolates have over time included serotypes O, A, C, Southern African Territories (SAT) 1 and SAT 2, mainly from livestock. SAT 3 has only been isolated in a few cases and only in African buffalos (Syncerus caffer). To investigate the presence of antibodies against FMDV serotypes in wildlife in Uganda, serological studies were performed on buffalo serum samples collected between 2001 and 2003. Thirty‐eight samples from African buffalos collected from Lake Mburo, Kidepo Valley, Murchison Falls and Queen Elizabeth National Parks were screened using Ceditest^® FMDV NS to detect antibodies against FMDV non‐structural proteins (NSP). The seroprevalence of antibodies against non‐structural proteins was 74%. To characterize FMDV antibodies, samples were selected and titrated using serotype‐specific solid phase blocking enzyme linked immunosorbent assay (ELISAs). High titres of antibodies (≥1 : 160) against FMDV serotypes SAT 1, SAT 2 and SAT 3 were identified. This study suggests that African buffalos in the different national parks in Uganda may play an important role in the epidemiology of SAT serotypes of FMDV.     

Opportunities for enhanced surveillance of foot‐and‐mouth disease in endemic settings using milk samples

Under‐reporting of foot‐and‐mouth disease (FMD) masks the true prevalence in parts of the world where the disease is endemic. Laboratory testing for the detection of FMD virus (FMDV) is usually reliant upon the collection of vesicular epithelium and fluid samples that can only be collected from acutely infected animals, and therefore animals with sub‐clinical infection may not be identified. Milk is a non‐invasive sample type routinely collected from dairy farms that has been utilized for surveillance of a number of other diseases. The aim of this study was to examine the application of milk as an alternative sample type for FMDV detection and typing, and to evaluate milk as a novel approach for targeted surveillance of FMD in East Africa. FMDV RNA was detected in 73/190 (38%) individual milk samples collected from naturally infected cattle in northern Tanzania. Furthermore, typing information by lineage‐specific rRT‐PCR assays was obtained for 58% of positive samples, and corresponded with the virus types identified during outbreak investigations in the study area. The VP1‐coding sequence data obtained from milk samples corresponded with the sequence data generated from paired epithelial samples collected from the same animal. This study demonstrates that milk represents a potentially valuable sample type for FMDV surveillance and might be used to overcome some of the existing biases of traditional surveillance methods. However, it is recommended that care is taken during sample collection and testing to minimize the likelihood of cross‐contamination. Such approaches could strengthen FMDV surveillance capabilities in East Africa, both at the individual animal and herd level.     

Serotype Specificity of Antibodies against Foot‐and‐Mouth Disease Virus in Cattle in Selected Districts in Uganda

Uganda had an unusually large number of foot‐and‐mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot‐and‐mouth disease virus (FMDV) by ELISA for antibodies against non‐structural proteins and structural proteins. Three hundred and forty‐nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non‐structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified antibodies. High prevalences of antibodies against non‐structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than against serotypes SAT 1, SAT 2 and SAT 3 in the sera investigated for serotype‐specific antibodies. Only FMDV serotype O virus was isolated from one probang sample. This study shows that the majority of the FMD outbreaks in 2006 in the region studied were caused by FMDV serotype O; however, there was also evidence of antibodies to both SAT 1 and SAT 3 in one outbreak in a herd inside Queen Elizabeth national park area.     

Foot‐and‐Mouth Disease Virus Serotype O Phylodynamics: Genetic Variability Associated with Epidemiological Factors in Pakistan

One of the most challenging aspects of foot‐and‐mouth disease (FMD) control is the high genetic variability of the FMD virus (FMDV). In endemic settings such as the Indian subcontinent, this variability has resulted in the emergence of pandemic strains that have spread widely and caused devastating outbreaks in disease‐free areas. In countries trying to control and eradicate FMD using vaccination strategies, the constantly evolving and wide diversity of field FMDV strains is an obstacle for identifying vaccine strains that are successful in conferring protection against infection with field viruses. Consequently, quantitative knowledge on the factors that are associated with variability of the FMDV is prerequisite for preventing and controlling FMD in the Indian subcontinent. A hierarchical linear model was used to assess the association between time, space, host species and the genetic variability of serotype O FMDV using viruses collected in Pakistan from 2005 to 2011. Significant (P < 0.05) amino acid and nucleotide variations were associated with spatial distance, but not with differences in host species, which is consistent with the frequent multi‐species infection of this serotype O FMDV. Results from this study will contribute to the understanding of FMDV variability and to the design of FMD control strategies in Pakistan. Viruses sequenced here also provide the earliest reported isolate from the Pan Asia II^ANT‐10 sublineage, which has caused several outbreaks in the Middle East and spread into Europe (Bulgaria) and Africa (Libya).