David J. Paton

Transmission Pathways of Foot-and-Mouth Disease Virus in the United Kingdom in 2007

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O1 BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.

Integrating genetic and epidemiological data to determine transmission pathways of foot-and-mouth disease virus

Estimating detailed transmission trees that reflect the relationships between infected individuals or populations during a disease outbreak often provides valuable insights into both the nature of disease transmission and the overall dynamics of the underlying epidemiological process. These trees may be based on epidemiological data that relate to the timing of infection and infectiousness, or genetic data that show the genetic relatedness of pathogens isolated from infected individuals. Genetic data are becoming increasingly important in the estimation of transmission trees of viral pathogens due to their inherently high mutation rate. Here, we propose a maximum-likelihood approach that allows epidemiological and genetic data to be combined within the same analysis to infer probable transmission trees. We apply this approach to data from 20 farms infected during the 2001 UK foot-and-mouth disease outbreak, using complete viral genome sequences from each infected farm and information on when farms were first estimated to have developed clinical disease and when livestock on these farms were culled. Incorporating known infection links due to animal movement prior to imposition of the national movement ban results in the reduction of the number of trees from 41 472 that are consistent with the genetic data to 1728, of which just 4 represent more than 95% of the total likelihood calculated using a model that accounts for the epidemiological data. These trees differ in several ways from those constructed prior to the availability of genetic data.

Production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus

This report describes the preparation of six monoclonal antibodies (MAbs) raised against a British isolate of porcine reproductive and respiratory syndrome virus (PRRSV), their characterization in terms of protein specificity and their reactivity with different PRRS viruses from Europe and the USA. Radioimmunoprecipitation and Western blotting studies of MAb reactivity with proteins from cell lysates of infected cells and purified virus revealed that four of the six MAbs (WBE1 and WBE4-6) precipitated a 15 kDa viral protein. Further studies using in vitro translated products of the Lelystad virus genome showed that this protein was the product of ORF7, the putative nucleocapsid protein. The specificity of another MAb, WBE2, was found to be for a 45 kDa protein, determined to be the product of ORF3 and demonstrated to be present in purified virion preparations. The protein specificity of the sixth MAb, WBE3 could not be determined. Thirty-three PRRSV isolates from Europe and the USA were grown in alveolar macrophages and examined by immunoperoxidase staining, using the panel of six MAbs. All European isolates were recognized by the four MAbs specific for the putative nucleocapsid, but the viruses showed different patterns of reactivity with WBE2 and WBE3. Furthermore, these two MAbs stained only a small proportion of the cells infected with certain isolates, suggesting that a single isolate may be antigenically heterogeneous. No MAbs bound to US isolates, indicating a consistent antigenic difference between the putative nucleocapsid of US and European isolates. Detergent extraction of cell lysate antigen abrogated the binding of WBE1-3, suggesting that the epitopes are conformation dependent.

Molecular Epidemiology of the Foot-and-Mouth Disease Virus Outbreak in the United Kingdom in 2001

ABSTRACT The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 × 10−5 per site per day (95% confidence interval [CI], 1.75 × 10−5 to 2.80 × 10−5) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.

Beyond the Consensus: Dissecting Within-Host Viral Population Diversity of Foot-and-Mouth Disease Virus by Using Next-Generation Genome Sequencing

ABSTRACT The diverse sequences of viral populations within individual hosts are the starting material for selection and subsequent evolution of RNA viruses such as foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the inoculum used to initiate experimental infection. Genomic sequences were determined in duplicate sequencing runs, and the consensus sequence of the inoculum determined by NGS was identical to that previously determined using the Sanger method. However, NGS revealed the fine polymorphic substructure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. Some of the higher-frequency polymorphisms identified encoded changes within codons associated with heparan sulfate binding and were present in both foot lesions, revealing intermediate stages in the evolution of a tissue culture-adapted virus replicating within a mammalian host. We identified 2,622, 1,434, and 1,703 polymorphisms in the inoculum and in the two foot lesions, respectively: most of the substitutions occurred in only a small fraction of the population and represented the progeny from recent cellular replication prior to onset of any selective pressures. We estimated the upper limit for the genome-wide mutation rate of the virus within a cell to be 7.8 × 10−4 per nucleotide. The greater depth of detection achieved by NGS demonstrates that this method is a powerful and valuable tool for the dissection of FMDV populations within hosts.

Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples

A lateral flow device (LFD) for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV) was developed using a monoclonal antibody (Mab 1F10) shown to be pan-reactive to FMDV strains of each serotype by ELISA. The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia (304 positive and 1003 negative samples) from suspected cases of vesicular disease collected from 86 countries between 1965 and 2008 and negative samples collected from healthy animals. The diagnostic sensitivity of the LFD for FMDV was similar at 84% compared to 85% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 99.9% for the ELISA. The device recognized FMDV strains of wide diversity of all seven serotypes but weaker reactions were often evident with those of type SAT 2, several viruses of which were not detected. Reactions with the viruses of swine vesicular disease and vesicular stomatitis that produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The test procedure was simple and rapid, and typically provided a result within 1-10min of sample addition. Simple homogenizers that could be used in field conditions for preparing epithelial suspensions were demonstrated to be effective for LFD application. These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease.

Multiple origins of foot-and-mouth disease virus serotype Asia 1 outbreaks, 2003-2007.

Viruses in 6 genetic groups have caused recent outbreaks in Asia.Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence.

Incursions of Foot-and-Mouth Disease Virus into Europe between 1985 and 2006

Foot-and-mouth disease (FMD) is one of the biggest threats to animal health in European countries. In the last 22 years (1985-2006), FMD has occurred 37 times in 14 European countries. Serotype O was most frequently involved in these outbreaks followed by A, C and Asia 1. Sometimes, epidemics were very limited and at other times, they were the cause of devastating economic losses. In most cases (22/37), the origin of the outbreaks could not be determined. For some of these outbreaks, however, routes of introduction and spread were identified through epidemiological inquiries. Moreover, in some cases, the origin of the virus was also traced by phylogenetic analysis of the partial or complete sequences of VP1 genes. Lessons learned from the outbreaks are still useful as most of the same risk factors persist. However, efforts made by FMD-free countries to help those where the disease is endemic are a valuable strategy for the reduction of the global risk. The present and the future potential sources of FMD infection need to be identified to best focus European efforts.

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.

Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza

The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of Infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.