Nicholas Juleff

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.

Relationship Between Clinical Signs and Transmission of an Infectious Disease and the Implications for Control

Livestock experiments provide precise parameters for incubation and infectious periods for foot-and-mouth disease virus. Control of many infectious diseases relies on the detection of clinical cases and the isolation, removal, or treatment of cases and their contacts. The success of such “reactive” strategies is influenced by the fraction of transmission occurring before signs appear. We performed experimental studies of foot-and-mouth disease transmission in cattle and estimated this fraction at less than half the value expected from detecting virus in body fluids, the standard proxy measure of infectiousness. This is because the infectious period is shorter (mean 1.7 days) than currently realized, and animals are not infectious until, on average, 0.5 days after clinical signs appear. These results imply that controversial preemptive control measures may be unnecessary; instead, efforts should be directed at early detection of infection and rapid intervention.

Foot-and-Mouth Disease Virus Persists in the Light Zone of Germinal Centres

Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV “carrier state” and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. Here we show by laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborate by in situ hybridization that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of naïve cattle. We propose that maintenance of non-replicating FMDV in these sites represents a source of persisting infectious virus and also contributes to the generation of long-lasting antibody responses against neutralising epitopes of the virus.

Foot-and-Mouth Disease Virus Can Induce a Specific and Rapid CD4+ T-Cell-Independent Neutralizing and Isotype Class-Switched Antibody Response in Naïve Cattle

ABSTRACT The role of T-lymphocyte subsets in recovery from foot-and-mouth disease virus (FMDV) infection in calves was investigated by administering subset-specific monoclonal antibodies. The depletion of circulating CD4+ or WC1+ γδ T cells was achieved for a period extending from before challenge to after resolution of viremia and peak clinical signs, whereas CD8+ cell depletion was only partial. The depletion of CD4+ cells was also confirmed by analysis of lymph node biopsy specimens 5 days postchallenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs, and immune responses following FMDV infection. Three of the four CD4+ T-cell-depleted calves failed to generate an antibody response to the nonstructural polyprotein 3ABC but generated a neutralizing antibody response similar to that in the controls, including rapid isotype switching to immunoglobulin G antibody. We conclude that antibody responses to sites on the surface of the virus capsid are T cell independent, whereas those directed against the nonstructural proteins are T cell dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1135-156 on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralizing sites on the virus capsid, are T cell dependent. The depletion of CD4+ T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. Overall, we conclude that CD4+ T-cell-independent antibody responses play a major role in the resolution of foot-and-mouth disease in cattle.

Characterization of Ovine Nectin-4, a Novel Peste des Petits Ruminants Virus Receptor

ABSTRACT Small ruminants infected with peste des petits ruminants virus exhibit lesions typical of epithelial infection and necrosis. However, the only established host receptor for this virus is the immune cell marker signaling lymphocyte activation molecule (SLAM). We have confirmed that the ovine Nectin-4 protein, when overexpressed in epithelial cells, permits efficient replication of PPRV. Furthermore, this gene was predominantly expressed in epithelial tissues and encoded by multiple haplotypes in sheep breeds from around the world.

Bovine Plasmacytoid Dendritic Cells Are the Major Source of Type I Interferon in Response to Foot-and-Mouth Disease Virus In Vitro and In Vivo

ABSTRACT Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3− CD14− CD21− CD11c− NK− TCRδ− CD4+ MHC II+ CD45RB+ CD172a+ CD32+). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.

Evolution of foot-and-mouth disease virus intra-sample sequence diversity during serial transmission in bovine hosts.

RNA virus populations within samples are highly heterogeneous, containing a large number of minority sequence variants which can potentially be transmitted to other susceptible hosts. Consequently, consensus genome sequences provide an incomplete picture of the within- and between-host viral evolutionary dynamics during transmission. Foot-and-mouth disease virus (FMDV) is an RNA virus that can spread from primary sites of replication, via the systemic circulation, to found distinct sites of local infection at epithelial surfaces. Viral evolution in these different tissues occurs independently, each of them potentially providing a source of virus to seed subsequent transmission events. This study employed the Illumina Genome Analyzer platform to sequence 18 FMDV samples collected from a chain of sequentially infected cattle. These data generated snap-shots of the evolving viral population structures within different animals and tissues. Analyses of the mutation spectra revealed polymorphisms at frequencies >0.5% at between 21 and 146 sites across the genome for these samples, while 13 sites acquired mutations in excess of consensus frequency (50%). Analysis of polymorphism frequency revealed that a number of minority variants were transmitted during host-to-host infection events, while the size of the intra-host founder populations appeared to be smaller. These data indicate that viral population complexity is influenced by small intra-host bottlenecks and relatively large inter-host bottlenecks. The dynamics of minority variants are consistent with the actions of genetic drift rather than strong selection. These results provide novel insights into the evolution of FMDV that can be applied to reconstruct both intra- and inter-host transmission routes.

CD4+ T-cell responses to foot-and-mouth disease virus in vaccinated cattle

We have performed a series of studies to investigate the role of CD4+ T-cells in the immune response to foot-and-mouth disease virus (FMDV) post-vaccination. Virus neutralizing antibody titres (VNT) in cattle vaccinated with killed FMD commercial vaccine were significantly reduced and class switching delayed as a consequence of rigorous in vivo CD4+ T-cell depletion. Further studies were performed to examine whether the magnitude of T-cell proliferative responses correlated with the antibody responses. FMD vaccination was found to induce T-cell proliferative responses, with CD4+ T-cells responding specifically to the FMDV antigen. In addition, gamma interferon (IFN-γ) was detected in the supernatant of FMDV antigen-stimulated PBMC and purified CD4+ T-cells from vaccinated cattle. Similarly, intracellular IFN-γ could be detected specifically in purified CD4+ T-cells after restimulation. It was not possible to correlate in vitro proliferative responses or IFN-γ production of PBMC with VNT, probably as a consequence of the induction of T-independent and T-dependent antibody responses and antigen non-specific T-cell responses. However, our studies demonstrate the importance of stimulating CD4+ T-cell responses for the induction of optimum antibody responses to FMD-killed vaccines.

Characterization of epitope-tagged foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.

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.