A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.

BACKGROUND: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. RESULTS: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. CONCLUSIONS: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

A thiazepino[4,5-a]benzimidazole derivative hampers the RNA replication of Eurasian serotypes of foot-and-mouth disease virus.

The stamping-out policy for the control of foot-and-mouth disease virus (FMDV) in countries that are free from FMD without vaccination has a dramatic socio-economic impact, huge animal welfare issues and may result in the loss of farm animal genetic resources. As an alternative to pre-emptive culling or emergency vaccination we further explore the possibility to use antiviral drugs in the event of an FMD outbreak. In the present study, we tested the in vitro cytotoxicity and anti-FMDV activity of 1,2,4,5-tetrahydro-[1,4]thiazepino[4,5-a]benzimidazole. The molecule was shown to inhibit the replication of reference strains of the Eurasian FMDV serotypes O, A, C and Asia but not the FMDV serotypes from the South African Territories (SAT) neither a related picornavirus, i.e. swine vesicular disease virus. The molecule can be added until 2h post inoculation in a 'single replication cycle experiment' without losing its antiviral activity. The genetic characterization of progressively selected resistant FMD viruses shows that the molecule presumably interacts with the non-structural 2C protein of FMDV. Further studies are required on the use of this molecule in vivo.

Accumulation of nucleotide substitutions occurring during experimental transmission of foot-and-mouth disease virus.

Analysis of full-genome sequences was previously used to trace the origin and transmission pathways of foot-and-mouth disease virus (FMDV) outbreaks in the UK in 2001 and 2007. Interpretation of these data was sometimes at variance with conventional epidemiological tracing, and was also used to predict the presence of undisclosed infected premises that were later discovered during serological surveillance. Here we report the genome changes associated with sequential passage of a highly BHK-21-cell-adapted (heparan sulphate-binding) strain of FMDV arising from two independent transmission chains in cattle. In vivo virus replication rapidly selected for a wild-type variant with an amino acid substitution at VP3(56). Full-genome sequence analysis clearly demonstrated sequence divergence during parallel passage. The genetic diversity generated over the course of infection and the rate at which these changes became fixed and were transmitted between cattle occurred at a rate sufficient to enable reliable tracing of transmission pathways at the level of the individual animal. However, tracing of transmission pathways was only clear when sequences from epithelial lesions were compared. Sequences derived from oesophageal-pharyngeal scrapings were problematic to interpret, with a varying number of ambiguities suggestive of a more diverse virus population. These findings will help to correctly interpret full-genome sequence analyses to resolve transmission pathways within future FMDV epidemics.

Genome sequences of SAT 2 foot-and-mouth disease viruses from Egypt and Palestinian Autonomous Territories (Gaza Strip).

Two foot-and-mouth disease virus (FMDV) genome sequences have been determined for isolates collected from recent field outbreaks in North Africa (Egypt) and the Middle East (Palestinian Autonomous Territories). These data represent the first examples of complete genomic sequences for the FMDV SAT 2 topotype VII, which is thought to be endemic in countries immediately to the south of the Sahara desert. Further studies are now urgently required to provide insights into the epidemiological links between these outbreaks and to define the pathogenicity of this emerging lineage.

Multiple introductions of serotype O foot-and-mouth disease viruses into East Asia in 2010-2011.

Foot-and-mouth disease virus (FMDV) is a highly contagious and genetically variable virus. Sporadic introductions of this virus into FMD-free countries may cause outbreaks with devastating consequences. In 2010 and 2011, incursions of the FMDV O/SEA/Mya-98 strain, normally restricted to countries in mainland Southeast Asia, caused extensive outbreaks across East Asia. In this study, 12 full genome FMDV sequences for representative samples collected from the People's Republic of China (PR China) including the Hong Kong Special Administrative Region (SAR), the Republic of Korea, the Democratic People's Republic of Korea, Japan, Mongolia and The Russian Federation were generated and compared with additional contemporary sequences from viruses within this lineage. These complete genomes were 8119 to 8193 nucleotides in length and differed at 1181 sites, sharing a nucleotide identity ≥ 91.0% and an amino acid identity ≥ 96.6%. An unexpected deletion of 70 nucleotides within the 5'-untranslated region which resulted in a shorter predicted RNA stem-loop for the S-fragment was revealed in two sequences from PR China and Hong Kong SAR and five additional related samples from the region. Statistical parsimony and Bayesian phylogenetic analysis provide evidence that these outbreaks in East Asia were generated by two independent introductions of the O/SEA/Mya-98 lineage sometime between August 2008 and March 2010. The rapid emergence of these viruses from Southeast Asia highlights the importance of adopting approaches to closely monitor the spread of this lineage that now poses a threat to livestock industries in other regions.

Southeast Asian foot-and-mouth disease viruses in Eastern Asia.

Foot-and-mouth disease (FMD) outbreaks recently affected 2 countries (Japan and South Korea) in eastern Asia that were free of FMD without vaccination. Analysis of viral protein 1 nucleotide sequences indicated that FMD serotype A and O viruses that caused these outbreaks originated in mainland Southeast Asia to which these viruses are endemic.

The impact of within-herd genetic variation upon inferred transmission trees for foot-and-mouth disease virus.

Full-genome sequences have been used to monitor the fine-scale dynamics of epidemics caused by RNA viruses. However, the ability of this approach to confidently reconstruct transmission trees is limited by the knowledge of the genetic diversity of viruses that exist within different epidemiological units. In order to address this question, this study investigated the variability of 45 foot-and-mouth disease virus (FMDV) genome sequences (from 33 animals) that were collected during 2007 from eight premises (10 different herds) in the United Kingdom. Bayesian and statistical parsimony analysis demonstrated that these sequences exhibited clustering which was consistent with a transmission scenario describing herd-to-herd spread of the virus. As an alternative to analysing all of the available samples in future epidemics, the impact of randomly selecting one sequence from each of these herds was used to assess cost-effective methods that might be used to infer transmission trees during FMD outbreaks. Using these approaches, 85% and 91% of the resulting topologies were either identical or differed by only one edge from a reference tree comprising all of the sequences generated within the outbreak. The sequence distances that accrued during sequential transmission events between epidemiological units was estimated to be 4.6 nucleotides, although the genetic variability between viruses recovered from chronic carrier animals was higher than between viruses from animals with acute-stage infection: an observation which poses challenges for the use of simple approaches to infer transmission trees. This study helps to develop strategies for sampling during FMD outbreaks, and provides data that will guide the development of further models to support control policies in the event of virus incursions into FMD free countries.

Genome Sequences of Foot-and-Mouth Disease Virus O/ME-SA/Ind-2001 Lineage from Outbreaks in Libya, Saudi Arabia, and Bhutan during 2013.

The complete genomes of foot-and-mouth disease (FMD) viruses recovered in Libya and Saudi Arabia in 2013 are described here. These viruses belong to an FMD virus lineage (Ind-2001, topotype Middle East-South Asia, serotype O) which is normally endemic in the Indian subcontinent. A contemporary virus sequence from Bhutan is also reported here.

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

Observing micro-evolutionary processes of viral populations at multiple scales.

Advances in sequencing technology coupled with new integrative approaches to data analysis provide a potentially transformative opportunity to use pathogen genome data to advance our understanding of transmission. However, to maximize the insights such genetic data can provide, we need to understand more about how the microevolution of pathogens is observed at different scales of biological organization. Here, we examine the evolutionary processes in foot-and-mouth disease virus observed at different scales, ranging from the tissue, animal, herd and region. At each scale, we observe analogous processes of population expansion, mutation and selection resulting in the accumulation of mutations over increasing time scales. While the current data are limited, rates of nucleotide substitution appear to be faster over individual-to-individual transmission events compared with those observed at a within-individual scale suggesting that viral population bottlenecks between individuals facilitate the fixation of polymorphisms. Longer-term rates of nucleotide substitution were found to be equivalent in individual-to-individual transmission compared with herd-to-herd transmission indicating that viral diversification at the herd level is not retained at a regional scale.

Development of a universal RT-PCR for amplifying and sequencing the leader and capsid-coding region of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly infectious viral disease of cloven-hoofed animals with debilitating and devastating consequences for livestock industries throughout the world. Key antigenic determinants of the causative agent, FMD virus (FMDV), reside within the surface-exposed proteins of the viral capsid. Therefore, characterization of the sequence that encodes the capsid (P1) is important for tracking the emergence or spread of FMD and for selection and development of new vaccines. Reliable methods to generate sequence for this region are challenging due to the high inter-serotypic variability between different strains of FMDV. This study describes the development and optimization of a novel, robust and universal RT-PCR method that may be used to amplify and sequence a 3kilobase (kb) fragment encompassing the leader proteinase (L) and capsid-coding portions (P1) of the FMDV genome. This new RT-PCR method was evaluated in two laboratories using RNA extracted from 134 clinical samples collected from different countries and representing a range of topotypes and lineages within each of the seven FMDV serotypes. Sequence analysis assisted in the reiterative design of primers that are suitable for routine sequencing of these RT-PCR fragments. Using this method, sequence analysis was undertaken for 49 FMD viruses collected from outbreaks in the field. This approach provides a robust tool that can be used for rapid antigenic characterization of FMDV and phylogenetic analyses and has utility for inclusion in laboratory response programs as an aid to vaccine matching or selection in the event of FMD outbreaks.

New technologies to diagnose and monitor infectious diseases of livestock: challenges for sub-Saharan Africa.

Using foot-and-mouth disease (FMD) as an example, this review describes new tools that can be used to detect and characterise livestock diseases. In recent years, molecular tests that can detect and characterise pathogens in a diverse range of sample types have revolutionised laboratory diagnostics. In addition to use in centralised laboratories, there are opportunities to locate diagnostic technologies close to the animals with suspected clinical signs. Work in this area has developed simple-to-use lateral-flow devices for the detection of FMD virus (FMDV), as well as new hardware platforms to allow molecular testing to be deployed into the field for use by non-specialists. Once FMDV has been detected, nucleotide sequencing is used to compare field strains with reference viruses. Transboundary movements of FMDV are routinely monitored using VP1 sequence data, while higher resolution transmission trees (at the farm-to-farm level) can be reconstructed using full-genome sequencing approaches. New technologies such as next-generation sequencing technologies are now being applied to dissect the viral sequence populations that exist within single samples. The driving force for the use of these technologies has largely been influenced by the priorities of developed countries with FMD-free (without vaccination) status. However, it is important to recognise that these approaches also show considerable promise for use in countries where FMD is endemic, although further modifications (such as sample archiving and strain and serotype characterisation) may be required to tailor these tests for use in these regions. Access to these new diagnostic and sequencing technologies in sub-Saharan Africa have the potential to provide novel insights into FMD epidemiology and will impact upon improved strategies for disease control.Effective control of infectious diseases is reliant upon accurate diagnosis of clinical cases using laboratory tests, together with an understanding of factors that impact upon the epidemiology of the infectious agent. A wide range of new diagnostic tools and nucleotide sequencing methods are used by international reference laboratories to detect and characterise the agents causing outbreaks of infectious diseases. In the past, high costs (initial capital expenses, as well as day-to-day maintenance and running costs) and complexity of the protocols used to perform some of these tests have limited the use of these methods in smaller laboratories. However, simpler and more cost-effective formats are now being developed that offer the prospect that these technologies will be even more widely deployed into laboratories particularly those in developing regions of the world such as sub-Saharan Africa.

Reconstruction of the transmission history of RNA virus outbreaks using full genome sequences: foot-and-mouth disease virus in Bulgaria in 2011.

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.