Interactions of foot-and-mouth disease virus with cells in organised lymphoid tissue influence innate and adaptive immune responses

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. The aim of this thesis was to study the interaction between FDMV and cells in lymphoid tissue in the natural bovine host, in order to improve our understanding of the protective immune response. Using laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborated by in situ hybridization, it is shown that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of naïve cattle. Maintenance of non-replicating FMDV in these sites may represent a source of persisting infectious virus and also contribute to the generation of long-lasting antibody responses against neutralising epitopes of the virus. The role of T-lymphocyte subsets in recovery from FMDV infection in calves was investigated by administering subset-specific mouse monoclonal antibodies. Depletion of circulating CD4+ or WC1+ γδ T cells was achieved for a period extending from before challenge to after resolution of viraemia and peak clinical signs, whereas CD8+ cell depletion was only partial. Depletion of CD4+ cells was also confirmed by analysis of lymph node biopsies 5 days post-challenge. 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 non-structural polyprotein 3ABC, but generated a neutralising antibody response similar to that in the controls, including rapid isotype switching to IgG antibody. These data suggest that antibody responses to sites on the surface of the virus capsid are T cell-independent whereas those directed against the non-structural 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 neutralising sites on the virus capsid, are T cell-dependent. Depletion of CD4+ T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. In conclusion, CD4+ T-cell-independent antibody responses play a major role in the resolution of primary infection with FMDV in cattle

Using Mathematical Modelling to Explore Hypotheses about the Role of Bovine Epithelium Structure in Foot-And-Mouth Disease Virus-Induced Cell Lysis.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. FMD virus (FMDV) shows a strong tropism for epithelial cells, and FMD is characterised by cell lysis and the development of vesicular lesions in certain epithelial tissues (for example, the tongue). By contrast, other epithelial tissues do not develop lesions, despite being sites of viral replication (for example, the dorsal soft palate). The reasons for this difference are poorly understood, but hypotheses are difficult to test experimentally. In order to identify the factors which drive cell lysis, and consequently determine the development of lesions, we developed a partial differential equation model of FMDV infection in bovine epithelial tissues and used it to explore a range of hypotheses about epithelium structure which could be driving differences in lytic behaviour observed in different tissues. Our results demonstrate that, based on current parameter estimates, epithelial tissue thickness and cell layer structure are unlikely to be determinants of FMDV-induced cell lysis. However, differences in receptor distribution or viral replication amongst cell layers could influence the development of lesions, but only if viral replication rates are much lower than current estimates.This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) [grant code: BBS/E/I/00001397], http://www.bbsrc.ac.uk/home/home.aspx. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from PLoS via http://dx.doi.org/10.1371/journal.pone.013857

An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein

Foot-and-mouth disease virus (FMDV) is one of the most extensively studied animal pathogens because it remains a major threat to livestock economies worldwide. However, the dynamics of FMDV infection are still poorly understood. The application of reverse genetics provides the opportunity to generate molecular tools to further dissect the FMDV life cycle. Here, we have used reverse genetics to determine the capsid packaging limitations for a selected insertion site in the FMDV genome. We show that exogenous RNA up to a defined length can be stably introduced into the FMDV genome, whereas larger insertions are excised by recombination events. This led us to construct a recombinant FMDV expressing the fluorescent marker protein, termed iLOV. Characterization of infectious iLOV-FMDV showed the virus has a plaque morphology and rate of growth similar to the parental virus. In addition, we show that cells infected with iLOV-FMDV are easily differentiated by flow cytometry using the inherent fluorescence of iLOV and that cells infected with iLOV-FMDV can be monitored in real-time with fluorescence microscopy. iLOV-FMDV therefore offers a unique tool to characterize FMDV infection in vitro, and its applications for in vivo studies are discussed

Cattle remain immunocompetent during the acute phase of foot-and-mouth disease virus infection

Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in naïve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3–4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models

Pervasive within-host recombination and epistasis as major determinants of the molecular evolution of the foot-and-mouth disease virus capsid

Although recombination is known to occur in foot-and-mouth disease virus (FMDV), it is considered only a minor determinant of virus sequence diversity. Analysis at phylogenetic scales shows inter-serotypic recombination events are rare, whereby recombination occurs almost exclusively in non-structural proteins. In this study we have estimated recombination rates within a natural host in an experimental setting. African buffaloes were inoculated with a SAT-1 FMDV strain containing two major viral sub-populations differing in their capsid sequence. This population structure enabled the detection of extensive within-host recombination in the genomic region coding for structural proteins and allowed recombination rates between the two sub-populations to be estimated. Quite surprisingly, the effective recombination rate in VP1 during the acute infection phase turns out to be about 0.1 per base per year, i.e. comparable to the mutation/substitution rate. Using a high-resolution map of effective within-host recombination in the capsid-coding region, we identified a linkage disequilibrium pattern in VP1 that is consistent with a mosaic structure with two main genetic blocks. Positive epistatic interactions between co-evolved variants appear to be present both within and between blocks. These interactions are due to intra-host selection both at the RNA and protein level. Overall our findings show that during FMDV co-infections by closely related strains, capsid-coding genes recombine within the host at a much higher rate than expected, despite the presence of strong constraints dictated by the capsid structure. Although these intra-host results are not immediately translatable to a phylogenetic setting, recombination and epistasis must play a major and so far underappreciated role in the molecular evolution of the virus at all scales.File. Supplementary methods and figures. Supplementary Information containing further details on statistical methods, data analysis and evolutionary consequences.Writing – review & editing: Luca Ferretti, Eva Pe´rez-Martı´n, Franc¸ois Maree, Bryan Charleston, Paolo Ribeca.The Pirbright Institute receives grant aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom (projects BB/E/I/00007035, BB/E/I/ 00007036, BB/E/I/00007032, BBS/E/I/00007039 and grant BB/L011085/1 as part of the joint USDANSF- NIH-BBSRC Ecology and Evolution of Infectious Diseases program).http://www.plospathogens.orgam2020Microbiology and Plant Patholog

Differential persistence of foot-and-mouth disease virus in African buffalo is related to virus virulence

Foot-and-mouth disease virus (FMDV) circulates as multiple serotypes and strains in many endemic regions. In particular the three Southern African Territories (SAT) serotypes are maintained effectively in their wildlife reservoir, the African buffalo, and individuals may harbour multiple SAT-serotypes for extended periods in the pharyngeal region. However the exact site and mechanism for persistence remain unclear. FMD in buffaloes offers a unique opportunity to study FMDV-persistence, as transmission from carrier ruminants has only convincingly been demonstrated for this species. Following co-infection of naïve African buffaloes with three SAT-serotypes isolated from field buffaloes; palatine tonsil swabs were the sample of choice for recovering infectious FMDV up to 400 days post infection (dpi). Post-mortem examination identified infectious virus for up to 185 dpi and viral genome up to 400 dpi in lymphoid tissue of the head and neck, mainly focussed in germinal centres. Interestingly viral persistence in vivo was not homogenous and the SAT-1 isolate persisted for longer than SAT-2 and SAT-3. Co-infection and passage of these SAT isolates in goat and buffalo cell lines demonstrated a direct correlation between persistence and cell killing capacity. These data suggest FMDV persistence occurs in the germinal centres of lymphoid tissue but the duration of persistence is related to virus replication and cell killing capacity.NJ was funded as a Wellcome Trust Intermediate Clinical Fellow and funding is acknowledged from the Biotechnology and Biological Sciences Research Council (BBS/E/I/00001523 and BBS/E/I/00001717).http://jvi.asm.org2016-11-30hb2016Microbiology and Plant Patholog

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

Demonstration of Co-Infection and Trans-Encapsidation of Viral RNA In Vitro Using Epitope-Tagged Foot-and-Mouth Disease Viruses

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is an economically devastating disease affecting several important livestock species. FMDV is antigenically diverse and exists as seven serotypes comprised of many strains which are poorly cross-neutralised by antibodies induced by infection or vaccination. Co-infection and recombination are important drivers of antigenic diversity, especially in regions where several serotypes co-circulate at high prevalence, and therefore experimental systems to study these events in vitro would be beneficial. Here we have utilised recombinant FMDVs containing an HA or a FLAG epitope tag within the VP1 capsid protein to investigate the products of co-infection in vitro. Co-infection with viruses from the same and from different serotypes was demonstrated by immunofluorescence microscopy and flow cytometry using anti-tag antibodies. FLAG-tagged VP1 and HA-tagged VP1 could be co-immunoprecipitated from co-infected cells, suggesting that newly synthesised capsids may contain VP1 proteins from both co-infecting viruses. Furthermore, we provide the first demonstration of trans-encapsidation of an FMDV genome into capsids comprised of proteins encoded by a co-infecting heterologous virus. This system provides a useful tool for investigating co-infection dynamics in vitro, particularly between closely related strains, and has the advantage that it does not depend upon the availability of strain-specific FMDV antibodies

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

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