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

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

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

<div><p>Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus <i>Nairovirus</i>. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus <i>in vivo</i>. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.</p></div

NSDV N protein distribution in lymph node, spleen, liver and kidney of infected sheep.

<p>Cryosections were prepared and stained as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124966#pone.0124966.g007" target="_blank">Fig 7</a>. Scale bars indicate 40 μm (A, B, D, E, G, H, J, K) or 10 μm (C, F, I).</p

Effect of NSDV infection on distribution of macrophages/monocytes in experimentally inoculated sheep.

<p>Cryosections were prepared as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124966#pone.0124966.g007" target="_blank">Fig 7</a> and stained with mouse monoclonal anti-calprotectin/L1 antibody (L1) and affinity-purified rabbit anti-NSDV N protein antibodies (NSDV N), followed by Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). DAPI was used as a counterstain (blue). Scale bars indicate 40 μm.</p

NSDV N protein distribution in caecum, duodenum and lung of infected sheep.

<p>Tissue samples were taken post-mortem from animals infected with the NSDVi isolate in a study previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124966#pone.0124966.ref018" target="_blank">18</a>], or from healthy animals that were not subject to any experimental procedures. Cryosections were prepared and sections were fixed and stained as described in Methods, using mouse monoclonal anti-collagen IV antibody (Coll) and affinity-purified rabbit anti-NSDV N protein antibodies (NSDV N), followed by AlexaFluor 488 goat anti-mouse IgG (green) and AlexaFluor 568 goat anti-rabbit IgG (red). DAPI was used as a counterstain (blue). Scale bars indicate 40 μm (A, B, D, E, G, H) or 10 μm (C, F, I).</p

Colocalisation of the N-terminal and the C-terminal part of the L protein in NSDV-infected cells.

<p>Vero cells were infected with the NSDVi isolate at a MOI of 1 TCID₅₀. After 16 h, cells were fixed using 4% PFA followed by ice cold methanol. <b>(A-H)</b>: Cells were stained sequentially with affinity-purified antibodies directed against the N-terminus of the L protein, Zenon AlexaFluor 488 (green) rabbit IgG labelling reagent (Fab:antibody ratio 5.6), and pre-made complex of affinity purified antibodies against the C-terminus of the L protein with Zenon AlexaFluor 594 (red) rabbit IgG labelling reagent (Fab:antibody ratio 3). <b>(I-P)</b>: Cells were stained sequentially with affinity purified anti-L(C-terminus) antibodies, Zenon Alexa Fluor 488 (green) rabbit IgG labelling reagent (Fab:antibody ratio 3.6), and then again with pre-made complex of affinity purified anti-L(C-terminus) antibodies with Zenon Alexa Fluor 594 (red) rabbit IgG labelling reagent (Fab:antibody ratio 3). Nuclei were counterstained using DAPI (blue). Representative focal planes from Z-stack series are shown. The ImarisColoc function of the Imaris x64 version 7.4.2 software was used to generate a colocalisation channel (D, H, L, P) from each 3D image, each of which was generated from eight focal planes through the thickness of an infected cell. Dashed white boxes in A-D and I-L indicate the area enlarged to show in E-H and M-P. Scale bars are shown.</p

Characterisation of NSDV core proteins in infected cells.

<p><b>(A)</b> Vero cells were infected with the NSDVi isolate at a MOI of 5 TCID₅₀ (NSDV) or left uninfected (uninf.). After 16 h, cells were harvested, lysed in sample buffer and proteins separated by SDS-PAGE; proteins were detected by Western blot using sera raised against the NSDV N protein, the C-terminus of the L protein or the N-terminus of the L protein, as indicated. <b>(B)</b> Sheep kidney epithelial cells (PO) or Vero cells were infected with the NSDVi isolate at a MOI of 0.3 TCID₅₀. After 16 h, cells were fixed using 4% PFA followed by ice cold methanol, and immunolabelled using sera raised against the NSDV N- or the C-terminus of the L protein followed by AlexaFluor-568 goat anti-rabbit IgG (red). DAPI was used as a counterstain (blue). Bars indicate 20 μm.</p

Quantitative analysis of colocalisation of the L protein N- and C-termini in infected cells.

<p>Three 3D images of infected cells, prepared as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124966#pone.0124966.g003" target="_blank">Fig 3</a> and each composed of eight focal slices, were analysed by the ImarisColoc function of the Imaris x64 version 7.4.2 software, using automatic threshold determination. <b>(A-D)</b> 2D plots of light intensity for each voxel in the 3D image for different pairs of channels for different pairs of antibodies: <b>(A)</b> plot of single channel against itself to illustrate theoretically perfect colocalisation; <b>(B)</b> plot of cytoplasmic L protein staining (green) vs nuclear DNA staining (blue) to illustrate perfect absence of colocalisation; <b>(C)</b> plot of actual perfect colocalisation, from staining infected cells with the same antibody (anti-L(CT)) labelled with two different fluorophores (Zenon 488 (green) or with Zenon 594 (red)); <b>(D)</b> plot of signal intensities given by anti-L(NT) and anti-L(CT). <b>(E)</b> Histogram showing average percentage of colocalisation (expressed as average “Pearson's coefficient in dataset volume”) between 488 and 594 signals from three analysed 3D images for each of control (infected cells stained only with anti-L(CT)) and experimental (infected cells co-stained with affinity purified anti-L(NT) and anti-L(CT)) samples. Error bars represent standard deviation.</p