Beyond the consensus: defining the significance of foot-and-mouth disease viral sequence diversity

Foot-and mouth disease is caused by a small virus (FMDV) which has an RNA genome. In common with most other RNA viruses, the replication machinery of FMDV makes errors when it copies the genome during replication. As a consequence, the virus evolves very rapidly and can quickly adapt to different environmental pressures. During the 2001 and 2007 outbreaks in the UK, we exploited these properties to finger-print FMDVs recovered from field samples to show how viruses collected on different farms were related to each other. These data provide valuable information to assist in epidemiological tracing and were used in real-time in 2007 to support Defra's control and eradication policy. It is likely that this type of analysis will be widely used to support any future incursions of FMD in the UK (or elsewhere in Europe). However, our current interpretation of these data is limited by our understanding of the fine-scale processes that underpin the genetic changes that are observed during transmission of the virus at the herd-to-herd (or animal-to-animal) level. The aim of this project is to use a 'next-generation' sequencing technology to reveal, for the first time, the complex mixture of viruses that are present within samples and are the starting material for fine-scale evolution of FMDV. We have conducted preliminary experiments to optimise and evaluate this method using a samples collected from a single cow that had been experimentally inoculated with FMDV. These data demonstrate that we are able to measure the frequency of even very rare genetic variants that exist in feet lesions from an infected animal. Many of these variants represent genetic intermediates that were previously undetected using conventional methods. We therefore, conclude that this new sequencing methodology is well-suited to revealing the fine substructure of complex viral populations and will be a valuable tool to quantify the high-resolution evolutionary dynamics of FMDV. During this project, we aim to extend this approach to generate data from a wider range of samples that have already been collected (and archived) from previous field outbreaks of FMD, and from controlled experimental transmission studies and develop models that quantify the effects of transmission on genetic diversity. In addition to improving our understanding of the way that FMDV evolves, we anticipate that the findings from this project will have broad application to the transmission biology of other acutely acting viral infections including viruses of human and veterinary importance such as classical swine fever, swine vesicular disease virus, rabies virus, influenza and corona-like (SARS) viruses.

RNA viruses such as foot-and-mouth disease virus (FMDV) evolve rapidly due to their high replication rate and poor proof-reading ability of their RNA-dependent RNA polymerase. These viruses are thought to exist as heterogeneous and complex populations comprising similar but non-identical genomes, but the evolutionary importance of this phenomenon remains unclear. Consensus sequencing identifies the predominant or major viral species present in a sample, but is uninformative about minority variants that may exist. Ignoring the polymorphic structure of viral populations reduces the reliability of approaches to use genome sequences to reconstruct the pattern of transmission between individuals. Cloning processes able to identify different sequence variants within a viral population are laborious and provide only a limited resolution of the mutant spectrum within a sample. This project aims to use 'next generation' sequencing technology to characterise how viral diversity within a host accumulates over the course of an acute infection of FMDV, and how much of this diversity is transmitted onward to susceptible individuals. Results from these transmission experiments will be compared to similar data generated over the course of outbreaks of FMD in the field. These new insights will inform and improve our tools that are used to trace FMDV during field outbreaks of disease by providing an improved understanding of the use of consensus sequences to reconstruct transmission trees, the development of new techniques for reconstructing transmission relationships from 'next generation' sequencing data, and new insights into how viral genetic differences accumulate with the number of transmission events. Our findings will likely have broad application to other important veterinary and human pathogens with similar replication strategies.

FMD is highly contagious and disease outbreaks are difficult to control. The exact mode of transmission between farms remains poorly understood and during the UK outbreak in 2001 the viral origin for many infected premises was attributed simply to 'local spread'. Here we address the important problem of how to use viral sequence data to infer the movement of virus between farms. As part of previously funded projects (BBSRC DTA and Defra) we have developed novel molecular tools that can be used to trace and map FMD virus spread between farms. These methods utilise full-genome sequences generated using 'conventional' Sanger methods and were used in real-time to link cases of FMD that occurred during 2007 in Surrey and Berkshire. These were widely used to demonstrate that the outbreaks were caused by a derivative of a FMDV reference strain (see independent reviews by Sir Iain Anderson, Professor Brian Spratt and the HSE). Analyses of these data revealed the most likely chain of transmission events, and predicted undisclosed infected premises prior to their discovery by serological surveillance. In addition to FMDV, prototypic techniques of this sort show real and immediate promise for use in managing outbreaks of many other viral pathogens with plastic RNA genomes (including important veterinary and human pathogens). However in order to maximize the robustness and confidence that can be placed in these inferences it is necessary to develop a more refined understanding of how the genetic signal in the data is generated, and transmitted between individuals - specifically in the case of FMD - between individuals within the same herd, and individuals in different herds. At the end of this project we will have: 1) Generated novel insights into fine-scale processes that drive the evolution of FMDV 2) Developed generic laboratory and analytical tools that can be used by others to study the evolution of RNA viruses 3) Developed new and improved viral tracing tools that can be used in real-time to support the FMD control and eradication programmes Methods and results generated through this project will increase our confidence in the use of this type of sequence data to support epidemiological investigations realising the potential of full-genome sequencing for analysis of future epidemics of FMD. While not a specific aim of this project, a closer understanding of how genetic diversity accumulates as virus is transmitted between individuals on different farms will lead to an increased real-time ability to infer when two farms are not linked by a direct transmission event, thus indicating the potential presence of other infectious farms that might be unknown to outbreak control operation.