Emergence of two novel sublineages Ind2001BD1 and Ind2001BD2 of foot‐and‐mouth disease virus serotype O in Bangladesh

Foot‐and‐mouth disease (FMD ) is endemic in Bangladesh, and the implementation of a control programme for this disease is at an early stage, according to the FAO ‐ and OIE ‐proposed Progressive Control Pathway for FMD (PCP ‐FMD ) Roadmap. To develop an effective control programme, understanding of foot‐and‐mouth disease virus (FMDV ) serotypes, even subtypes within the serotypes is essential. The present investigation aims at viral VP 1 coding region sequence‐based analysis of FMD samples collected from 34 FMD outbreaks during 2012–2016 in Bangladesh. Foot‐and‐mouth disease virus (FMDV ) serotype O was responsible for 82% of the outbreaks in Bangladesh, showing its dominance over serotype A and Asia1. The VP 1 phylogeny revealed the emergence of two novel sublineages of serotype O, named as Ind2001BD 1 and Ind2001BD 2, within the Ind2001 lineage along with the circulation of Ind2001d sublineage in Bangladesh, which was further supported by the multidimensional scaling with distinct clusters for each sublineage. The novel sublineages had evident genetic variability with other established sublineages within Ind2001 lineage. Ten mutations with three or more amino acid variations were detected within B‐C loop, G‐H loop and C‐terminal region of the VP 1 protein of FMDV serotype O viruses isolated exclusively from Bangladesh. Furthermore, two amino acid substitutions at positions 197 and 198 within the VP 1 C‐terminal region are unique to the novel sublineages. The existence of widespread genetic variations among circulatory FMDV serotype O viruses makes the FMD control programme complex in Bangladesh. Adequate epidemiological data, disease reporting, animal movement control, appropriate vaccination and above all stringent policies of the government are necessary to combat FMD in Bangladesh.     

Review of the Global Distribution of Foot‐and‐Mouth Disease Virus from 2007 to 2014

Foot‐and‐mouth disease (FMD) virus affects livestock worldwide. There are seven different serotypes, each with a diversity of topotypes, genetic lineages and strains. Some lineages have different properties that may contribute to sporadic spread beyond their recognized endemic areas. The objective of this study was to review the most significant FMD epidemiological events that took place worldwide between 2007 and 2014. Severe epidemics were caused by FMD virus (FMDV) lineage O/Asia/Mya‐98 in Japan and South Korea in 2010, both previously free of disease. In India, where FMD is endemic, the most important event was the re‐emergence of lineage O/ME‐SA/Ind‐2001 in 2008. Notably, this lineage, normally restricted to India, Bangladesh, Nepal and Bhutan, was also found in Saudi Arabia and Libya in 2013 and has caused several outbreaks in Tunisia and Algeria in 2014–2015. In January 2011, FMDV‐positive wild boars were found in Bulgaria, where the disease last occurred in 1996, followed by 12 outbreaks in livestock infected with FMDV O/ME‐SA/PanAsia2. In 2012, FMDV SAT2 caused outbreaks in Egypt and the Palestinian Autonomous Territories. Another significant event was the emergence of FMDV Asia1 Sindh‐08 in the Middle East. In South America, one outbreak of FMDV serotype O, topotype Euro‐SA was reported in Paraguay in 2011, which was recognized as FMD‐free with vaccination at the time. Lessons learned from past events, point out the need for an integrated strategy that comprises coordinated global and regional efforts for FMDV control and surveillance. Specific local characteristics related to host, environment and virus that condition FMD occurrence should be carefully considered and incorporated to adapt appropriate strategies into local plans. In this review, we compiled relevant epidemiological FMD events to provide a global overview of the current situation. We further discussed current challenges present in different FMD areas.     

Emergence and Distribution of Foot‐and‐Mouth Disease Virus Serotype A and O in Bangladesh

Foot‐and‐mouth disease (FMD) is endemic in Bangladesh and is predominantly due to FMDV serotype O. In 2012, FMD outbreaks were identified in five different districts of Bangladesh. Of 56 symptomatic cattle epithelial tissue samples, diagnostic PCR assay based on 5′‐URT detected 38 FMDV infections. Viral genotyping targeting VP1‐encoding region confirmed emergence of two distinct serotypes, A and O with an abundance of serotype A in Chittagong and Gazipur districts and serotype O in Pabna and Faridpur. Only single lineage of both A and O was retrieved from samples of five different regions. Sequencing and phylogenetic analysis of VP1 sequences revealed that serotype O sequences were closely related to the Ind 2001 sublineage of Middle East–South Asia (ME‐SA) topotype that was previously circulating in Bangladesh, and serotype A sequences belonging to the genotype VII that was dominant in India during the last decade. The results suggest that extensive cross‐border animal movement from neighbouring countries is the most likely source of FMDV serotypes in Bangladesh.     

Emergence of an exotic strain of serotype O foot‐and‐mouth disease virus O/ME‐SA/Ind‐2001d in South‐East Asia in 2015

The O/Middle East‐South Asia (ME ‐SA )/Ind‐2001 lineage of foot‐and‐mouth disease virus (FMDV ) is endemic in the Indian subcontinent and has been reported in the Middle East and North Africa, but it had not been detected in South‐East Asia (SEA ) before 2015. This study reports the recent incursions of this viral lineage into SEA , which caused outbreaks in Vientiane Capital of Lao People's Democratic Republic (PDR ) in April 2015, in Dak Nong, Dak Lak and Ninh Thuan Provinces of Vietnam from May to October 2015, and in Rakhine State of Myanmar in October 2015. Disease investigations were conducted during the outbreaks and followed up after laboratory results confirmed the involvement of FMDV O/ME ‐SA /Ind‐2001 sublineage d (O/ME‐SA/Ind‐2001d). Affected host species included cattle, buffalo and pig, and all the outbreaks resolved within 2 months. Animals with clinical signs were separated, and affected premises were disinfected. However, strict movement restrictions were not enforced, and emergency vaccinations were only implemented in Vientiane Capital of Lao PDR and Dak Nong and Ninh Thuan Provinces of Vietnam. Clinical samples were collected from each outbreak and examined by nucleotide sequencing of the FMDV viral protein 1 coding region. Sequence analysis revealed that the O/ME ‐SA /Ind‐2001d isolates from Lao PDR and Vietnam were closely related to each other and similar to viruses previously circulating in India in 2013. Viruses collected from Myanmar were divergent from viruses of the same sublineage recovered from Lao PDR and Vietnam but were closely related to viruses present in Bangladesh in 2015. These findings imply that at least two independent introductions of O/ME ‐SA /Ind‐2001d into SEA have occurred. Our study highlights the transboundary nature of foot‐and‐mouth disease (FMD ) and reinforces the importance of improved FMD surveillance and promotion of safer cross‐border trade in SEA to control the risk of introduction and spread of exotic FMDV strains.     

A Survey on the Frequency of Foot‐and‐Mouth Disease Virus Carriers in Cattle in North–East of Iran by RT‐PCR: Implications for Revising Disease Control Strategy

Foot‐and‐mouth disease (FMD) is endemic in Iran. It is essential to timely evaluate the current disease control programme in Iran. Here, we report the frequency of FMD virus (FMDV) carrier state in cattle slaughtered in Mashhad abattoir, Mashhad, Khorasan Razavi, north–east of Iran, which contains long common borders with Afghanistan and Turkmenistan. Soft palate samples were collected immediately after slaughter for the detection of FMDV by RT‐PCR. The results show that 37.7% of cattle (96 of 255) were carriers of the virus. Among positive samples (96), 58 (60.4%) belonged to serotype O. No evidence was detected for the presence of Asia 1 and A serotypes. Nucleotide sequencing and phylogenic dendogram showed close similarity and common lineage between our samples and viruses isolated in Pakistan. With an approximate more than 80% of cattle population vaccination coverage such a high rate of carrier state may show an extensive FMDV exposure. Therefore, limiting control programmes to timely prophylactic vaccination may be insufficient. This is also true when meat market instabilities act as a temptation to import livestock, legally or illegally, through the eastern frontiers. It is recommended to change the current prophylactic vaccination strategy to a well‐developed regional control programme, with close monitoring of animal movement through eastern frontiers, supported by government commitment and educational programmes. Timely estimation of the frequency of carrier state both in cattle and small ruminants is also advocated as a gauge to monitor the virus status in the region.     

Genetic Characterization of Serotypes A and Asia‐1 Foot‐and‐mouth Disease Viruses in Balochistan,Pakistan, in 2011

This study reports characterization of foot‐and‐mouth disease virus (FMDV) in samples collected from Balochistan, Pakistan. FMDV was detected by pan‐FMDV real‐time RT‐PCR in 31 samples (epithelial and oral swabs) collected in 2011 from clinical suspect cases. Of these, 29 samples were serotyped by serotype‐specific real‐time RT‐PCR assays and were confirmed by sequencing the VP1 coding region. Sixteen samples were found positive for serotype A and eight for serotype Asia‐1, whereas five samples were found positive for both serotypes A and Asia‐1. Two serotype A positive samples were found positive for two different strains of serotype A FMDV each. Phylogenetic analyses of serotype A FMDVs showed circulation of at least three different sublineages within the A‐Iran05 lineage. These included two earlier reported sublineages, A‐Iran05^HER−10 and A‐Iran05^FAR−11, and a new sublineage, designated here as A‐Iran05^BAL−11. This shows that viruses belonging to the A‐Iran05 lineage are continuously evolving in the region. Viruses belonging to the A‐Iran05^FAR−11 sublineage showed close identity with the viruses circulating in 2009 in Pakistan and Afghanistan. However, viruses belonging to the A‐Iran05^HER−10 detected in Balochistan, Pakistan, showed close identity with the viruses circulating in Kyrgyzstan, Iran and Kazakhstan in 2011 and 2012, showing that viruses responsible for outbreak in these countries have a common origin. Serotype Asia‐1 FMDVs reported in this study all belonged to the earlier reported Group‐VII (Sindh‐08), which is currently a dominant strain in the West Eurasian region. Detection of two different serotypes of FMDV or/and two different strains of the same serotype in one animal/sample shows complexity in occurrence of FMD in the region.     

Foot‐and‐mouth disease outbreaks due to an exotic serotype Asia 1 virus in Myanmar in 2017

In January 2017, two villages located in Rakhine State of Myanmar reported clinical signs in cattle suggestive of foot‐and‐mouth disease virus (FMDV) infection. Laboratory analysis identified the outbreak virus as FMDV serotype Asia 1, which represented the first detection of this serotype in Myanmar since 2005 and in the region of South‐East Asia (SEA) since 2007. Genetic analysis revealed that the outbreak virus was different from historical viruses from Myanmar and was more closely related to viruses circulating in Bangladesh and India during 2012–2013, indicating that a novel viral introduction had occurred. The precise origin of the outbreaks was not clear, but frequent informal livestock trade with South Asia was reported. Responses to the outbreaks involved disinfection, quarantine and animal movement restrictions; no further outbreaks were detected under the present passive surveillance system. Detection of serotype Asia 1 highlights the complex and dynamic nature of FMDV in SEA. Active surveillance is needed to assess the extent and distribution of this exotic Asia 1 strain and continued vigilance to timely detect the occurrence of emerging and re‐emerging FMDV strains is essential.     

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.     

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.     

Genetic diversity and comparison of diagnostic tests for characterization of foot‐and‐mouth disease virus strains from Pakistan 2008–2012

We report the laboratory analysis of 125 clinical samples from suspected cases of foot‐and‐mouth disease (FMD ) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT ‐PCR , of which 88 were also found to contain infectious FMD virus (FMDV ) by virus isolation (VI ), with strong correlation between these tests (κ = 0.96). Samples that were VI ‐positive were serotyped by antigen detection ELISA (Ag‐ELISA ) and VP 1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n  = 13), O (n  = 36) and Asia‐1 (n  = 41), including three samples from which both serotypes Asia‐1 and O were detected. Serotype A viruses were classified within three different Iran‐05 sublineages: HER ‐10, FAR ‐11 and ESF ‐10. All serotype Asia‐1 were within Group VII (Sindh‐08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia‐2 within two different sublineages: ANT ‐10 and BAL ‐09. Using VP 1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag‐ELISA to correctly determine serotype was 74%, and serotype‐specific sensitivity was 8% for serotype A, 88% for Asia‐1 and 89% for O. Serotype‐specific specificity was 100% for serotype A, 93% for Asia‐1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag‐ELISA . This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV .     

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).     

Analytical Epidemiology of Genomic Variation among Pan Asia Strains of Foot‐and‐Mouth Disease Virus

Genetic data from field isolates of foot‐and‐mouth disease virus (FMDV) have been used to trace the source of recent outbreaks of FMD, to design better vaccines and diagnostic tests for FMDV, and to make conclusions regarding the general variability in the FMDV genome. Though epidemiologic data associated with FMDV isolates are available, these data have been used rarely to explore possible associations of epidemiologic factors with evolution or variation of the FMDV genome. In this study, factors associated with variation in the primary immunogenic peptide gene of FMDV (VP1), for a sample of 147 serotype O, Pan Asia strain sequences were explored using traditional analytical epidemiologic methods: logistic regression and multinomial‐response logistic regression. Hypothesized factors included host type (bovine, ovine, buffalo, or porcine) and geographical region (Middle East, South Asia, East Asia, Southeast Asia, and Europe). Results of two regression analyses suggest that host type and region, considered to be possible surrogates for host management, may be associated with selection in the VP1 amino acid sequence in FMDV. For example, isolates from cattle and sheep in South Asia appear to converge with a proposed ancestor sequence, whereas isolates from the same species in the Middle East and Southeast Asia appear to diverge. The methods demonstrated here could be used on a more detailed dataset to explore the selective pressure of host immunity on the evolution of FMDV antigens in an endemic setting. More broadly, epidemiologic methods could be applied extensively to molecular data to explore the causes of genomic variation in disease‐causing organisms.     

Characterization of Foot‐and‐Mouth Disease Viruses Collected in Nigeria Between 2007 and 2014: Evidence for Epidemiological Links Between West and East Africa

This study describes the molecular characterization of 47 foot‐and‐mouth disease (FMD) viruses recovered from field outbreaks in Nigeria between 2007 and 2014. Antigen ELISA of viral isolates was used to identify FMD virus serotypes O, A and SAT 2. Phylogenetic analyses of VP1 nucleotide sequences provide evidence for the presence of multiple sublineages of serotype SAT 2, and O/EAST AFRICA 3 (EA‐3) and O/WEST AFRICA topotypes in the country. In contrast, for serotype A, a single monophyletic cluster of viruses has persisted within Nigeria (2009–2013). These results demonstrate the close genetic relatedness of viruses in Nigeria to those from other African countries, including the first formal characterization of serotype O/EA‐3 viruses in Nigeria. The introductions and persistence of certain viral lineages in Nigeria may reflect transmission patterns via nomadic pastoralism and animal trade. Continuous monitoring of field outbreaks is necessary to dissect the complexity of FMD epidemiology in sub‐Saharan Africa.     

Serotype Diversity of Foot‐and‐Mouth‐Disease Virus in Livestock without History of Vaccination in the Far North Region of Cameroon

Little information is available about the natural cycle of foot‐and‐mouth disease (FMD) in the absence of control measures such as vaccination. Cameroon presents a unique opportunity for epidemiological studies because FMD vaccination is not practiced. We carried out a prospective study including serological, antigenic and genetic aspects of FMD virus (FMDV) infections among different livestock production systems in the Far North of Cameroon to gain insight into the natural ecology of the virus. We found serological evidence of FMDV infection in over 75% of the animals sampled with no significant differences of prevalence observed among the sampled groups (i.e. market, sedentary, transboundary trade and mobile). We also found antibodies reactive to five of the seven FMDV serotypes (A, O, SAT1, SAT2 and SAT3) among the animals sampled. Finally, we were able to genetically characterize viruses obtained from clinical and subclinical FMD infections in Cameroon. Serotype O viruses grouped into two topotypes (West and East Africa). SAT2 viruses grouped with viruses from Central and Northern Africa, notably within the sublineage causing the large epidemic in Northern Africa in 2012, suggesting a common origin for these viruses. This research will guide future interventions for the control of FMD such as improved diagnostics, guidance for vaccine formulation and epidemiological understanding in support of the progressive control of FMD in Cameroon.     

Characterization of SAT2 foot‐and‐mouth disease 2013/2014 outbreak viruses at the wildlife–livestock interface in South Africa

The Southern African Territories (SAT)‐type foot‐and‐mouth disease viruses (FMDV) are endemic to the greater Kruger National Park (KNP) area in South Africa, where they are maintained through persistent infections in African buffalo. The occurrence of FMDV within the Greater KNP area constitutes a continual threat to the livestock industry. To expand on knowledge of FMDV diversity, the genetic and antigenic relatedness of SAT2‐type viruses isolated from cattle during a FMD outbreak in Mpumalanga Province in 2013 and 2014 were investigated. Cattle from twelve diptanks tested positive on polymerase chain reaction (PCR), and molecular epidemiological relationships of the viruses were determined by VP1 sequencing. Phylogenetic analysis of the SAT2 viruses from the FMD outbreak in Mpumalanga in 2013/2014 revealed their genetic relatedness to other SAT2 isolates from topotype I (South Africa, Zimbabwe and Mozambique), albeit genetically distinct from previous South African outbreak viruses (2011 and 2012) from the same topotype. The fifteen SAT2 field isolates clustered into a novel genotype with ≥98.7% nucleotide identity. High neutralization antibody titres were observed for four 2013/2014 outbreak viruses tested against the SAT2 reference antisera representative of viruses isolated from cattle and buffalo from South Africa (topotype I) and Zimbabwe (topotype II). Comparison of the antigenic relationship (r₁ values) of the outbreak viruses with reference antisera indicated a good vaccine match with 90% of r₁ values > 0.3. The r₁ values for the 2013/2014 outbreak viruses were 0.4 and above for the three South African vaccine/reference strains. These results confirm the presence of genetic and antigenic variability in SAT2 viruses and suggest the emergence of new variants at the wildlife–livestock interface in South Africa. Continuous characterization of field viruses should be performed to identify new virus strains as epidemiological surveillance to improve vaccination efforts.     

Status of Foot‐and‐mouth Disease in India

Foot‐and‐mouth disease (FMD) is endemic in India and causes severe economic loss. Status of FMD in the country for five fiscal years is presented. Outbreaks were more in number in 2007–2008 than 2010–2011. Three serotypes of FMD virus (O, A and Asia1) are prevalent. Serotype O was responsible for 80% of the confirmed outbreaks/cases, whereas Asia1 and A caused 12% and 8%, respectively. Geographical region‐wise assessment indicated varying prevalence rate in different regions viz; 43% in Eastern region, 31.5% in Southern region, 11.6% in North‐eastern region, 5% Central region, 4.4% Western region and 4% in Northern region. Highest number of outbreaks/cases was recorded in the month of September and lowest in June. Emergence and re‐emergence of different genotypes/lineages within the serotypes were evident in real‐time investigation carried out from time to time. Continues antigenic divergence in serotype A resulted in change in the vaccine strain in 2009. As on date, all genetic diversity within the serotypes is well tolerated by the vaccine strains. Unrestricted animal movements in the country play a major role in the spread of FMD.