Deliverables
Background: Although the pathogenesis of foot-and-mouth disease (FMD) has been extensively investigated previously, relatively little data are available about the localisation of FMD virus (FMDV) in vivo. Accordingly, the aim was to increase our understanding of the localisation of FMDV and the early events in infection. To this end, the cells in the ruminant upper respiratory tract infected with FMDV were identified, during acute and persistent infection. Laser Microdissection (LMD) linked with real-time PCR (LMD/qRT-PCR) were employed to estimate levels of FMDV genomes at specific sites during infection.
Results: During the acute phase of FMD, infection was detected in the epithelium of tongue, foot and pharyngeal tissues. FMDV detection was very strong in the germinativum layer and concentrated in the cytoplasm of the cell. In the spinosum layer, the FMDV detection is more diffuse and observed throughout this layer of the lesion. No positive signal was found in the corneum layer. Mucus gland and pharyngeal lymphoid nodules nearby the epithelium were also positive for FMDV.
During the persistent stage, except in the epithelial cells of the soft palate and nasopharynx, FMDV was also found in the germinativum layer of the epithelium of the dorsal side soft palate and of the nasopharynx. The virus was also present in the lymph nodules and mucus glands of the dorsal soft palate and nasopharynx.
The LMD/qRT-PCR assay generated quantitative data on viral RNA levels in different cellular layers of epithelium during the infection, and allowed identification of the early localisation sites of the virus during the establishment of infection.. The highest levels of FMDV were detected within the stratum spinosum during the acute stage of disease. Within the foot epithelia, the first site of infection was the stratum basale, which is on the basement membrane; within the soft palate the primary cell type of infection is the spinosum cells of the dorsal surface.
These data provide the essential basis for the understanding of development of FMD pathogenesis and carrier status, and provide for future detailed analyses oof infected and carrier animals.
Background: In order to determine the importance of particular integrins - and indeed heparan sulphate entities on cell surfaces - for infection of host cells by FMDV, it is necessary to possess the appropriate viral tools. To this end, it is necessary to construct FMD viruses with mutations in the RGD receptor binding site within the GH loop of the viral VP1. Accordingly, the aim was to generate FMDV viruses with mutations in the amino acids flanking this RGD receptor binding site, for application in studies on integrin receptor binding specificity of the virus.
Results: Recombinant FMD viruses were generated from plasmid DNA. The integrin binding loop (the G-H loop of the viral VP1) was sequenced for these rescued viruses. This allowed description of the integrin expressed on porcine and bovine cells targeted by FMDV. Consequently, the integrin specificity of the recombinant FMD viruses was established with cells derived from the natural hosts for the virus.
This integrin specificity was also correlated with infectivity in vivo. RNA inoculation experiments in suckling mice identified the sequence requirements in the VP1 G-H loop for viral entry. Certain amino acid positions were seen to be particularly relevant for interaction with the integrins, while others were dispensable. Actually, in some cases the capsid sequence changed to accommodate the introduced alterations in the G-H loop. These changes introduced into the G-H loop modified virus infectivity in cultured cells and in animals the level of modified infectivity was dependent on the particular change introduced into the G-H loop.
This work has allowed conclusions to be drawn on integrin usage by FMDV, as well as modulation of virus-integrin interaction, in terms of virus infection in cell culture and in vivo.
Background: Dendritic cells (DC) play a central role in immune response development and are an important target for vaccines. These cells are critical for initiating and controlling both innate and adaptive (specific) immune responses. In this sense, efficacious vaccines must efficiently interact with the DC, leading to the correct processing of the vaccine antigen by the DC. Only then will an efficient stimulation of the appropriate immune defence development occur. It is therefore important to consider the manner by which FMDV interacts with DC, and the particular efficiency when heparan sulphate (HS) binding capacity is present. To this end, it is important to understand the immunological consequences of infection of DC with HS- and non-HS-variant viruses. Accordingly, the aim was to determine the immunological consequences of FMDV interaction with monocytic cells.
- FMDV interaction with myeloid DC
FMDV antigen-positive DC clearly process the virus antigen, being efficient at inducing antigen specific responses by T lymphocytes isolated from immune animals. Moreover, antigen-specific B lymphocytes were also induced by FMDV-carrying DC to produce specific antibody this was dependent on the presence of DC carrying FMDV antigen. The additional presence of the CD6+ T cells augmented the responsiveness of the B lymphocytes, but this could be replaced by IL-2 plus IL-4.
DC, which had endocytosed-non-HS virus, would also induce antigen-specific T and B cell activities, showing that antigen had indeed been internalised and was probably processed too rapidly to be visualised. Nevertheless, the efficiency of inducing T and B cell activities was higher with the HS-binding virus, reflecting the greater retention of this type of antigen in the DC. That is, the DC are showing evidence for a more efficient uptake and retention of HS-binding virus (as found in vaccine preparations) for processing and presentation to the T and B lymphocytes. This was observed with both live and inactivated virus. Consequently, the presence of the FMDV in DC does not impair their ability to induce T and B lymphocyte activity. On the contrary, efficient uptake and retention of the virus by DC is essential.
- FMDV interaction with plasmacytoid DC (natural interferon-producing cells)
FMDV (live or inactivated) did not induce IFNa production by the natural interferon-producing cells (NIPC), nor did it interfere with the capacity of known stimuli of NIPC to induce interferon production. Interestingly, when live FMDV was complexed with immune IgG, there was a clear ability to induce interferon production by NIPC.
This was dependent on the internalised virus producing a dsRNA intermediate, although the replicative cycle was abortive.
This is important for understanding the importance of these innate defenders during the development of specific immunity, and would clearly present a critical defence at times when antibody development was only beginning. That is, the roles played by NIPC is of particular relevance early post-vaccination, and would be of high value under conditions of emergency vaccination when rapid protection is required. The NIPC would respond to immune complexes, even when the virus infectivity was not neutralised. The production of the interferon would enhance the immune responses, but also assist the anti-viral defence, aiding the host to resist the virus infection until such times as the antibody levels became fully protective. This could explain the means by which vaccinates can be protected early post-vaccination when antibody levels are still low.
Background: FMDV is known to interact with macrophages, but nothing is known about the interaction of the virus with dendritic cells (DC). This is important considering the critical role played by DC in initiating and controlling both innate and adaptive (specific) immune responses.
Moreover, vaccine design requires that the virus (inactivated viral antigen) interact with the DC in an efficient manner such that the antigen is correctly processed by the DC leading to stimulation of immune defence development. Incorrect interaction of the virus antigen with the DC can lad to destruction of the antigen without stimulation of the immune defences. Accordingly, the aim was to determine the capacity of FMDV to bind to and interact with DC of a natural host the pig.
Results: FMDV binds to monocytes (Mo), macrophages (MF), and dendritic cells (DC) but no virus replication occurs. This interaction was particularly efficient when the virus could employ heparan sulphate (HS) entities on the cell surface, but not when only integrins were available. When HS binding and non-HS-binding virus variants were compared, the former were the more efficiently internalised by the DC. Moreover, DC does not express the avb6 integrin demonstrated in this project to be the main integrin receptor for FMDV.
This importance of HS binding for FMDV interaction with DC was confirmed using dispirotripiperazine (DSTP), a blocker of HS binding. The DSTP blocked the uptake of the HS-binding viruses, demonstrating that these viruses did indeed employ the HS structures on the DC surface for binding and subsequent internalisation by the cells. With the non-HS viruses, these were rapidly internalised by the DC, with a rapid processing rendering antigen detection difficult. Although HS was not employed, and avb6 integrin was not present on the DC, it is likely that these non-HS binding viruses were employing other integrin structures for binding to the DC. None of the integrins currently detectable on porcine cells were involved in this process.
HS-binding virus is internalised by DC within 5 min, becoming distributed within the peripheral cytoplasm within 1hr, and still detectable after 4 hr. This is unaffected by bafilomycin treatment of the cells (inhibits endosomal acidification processes). With the non-HS virus in the presence of bafilomycin, the virus remained around the periphery without being internalised. In the absence of bafilomycin, no virus was detectable. These results suggest that the HS-binding virus enters DCs by an endosomal-independent manner, whereas non-HS virus enters via processes relying on rapid endosomal acidification.
DC can also be transfected with RNA derived from FMDV infectious DNA clones. Low levels of antigen and infectious virus can be detected at 24 hr p.i., but this is processed by the DC within the next 24 hr to become undetectable.
These results are demonstrating that the presence of heparin sulphate binding capacity in an FMDV preparation aids the interaction of the virus with DC, giving HS-binding virus and advantage over non-HS binding virus. This is particularly important considering that the DC do not possess the avb6 integrin demonstrated in this project to be the main integrin receptor for FMDV. It is also important for vaccine manufacturers when their vaccine virus has been cell culture passaged and therefore carrying this HS binding capacity.
The effort towards this TIP also demonstrated that the DC can accommodate RNA transfection, which might prove a usual means of vaccination in the future.
Background: FMDV has been reported to use a number of av integrins as receptors to initiate infection of cells in culture. However, the contribution of specific integrins to FMDV tropism and pathogenesis were unknown. Accordingly, the aim was to characterise integrin expression in the upper respiratory tract during infection with FMDV. In addition, stable cell lines expressing one of the known integrin receptors for FMDV were generated to facilitate the detailed analysis of the roles played by integrins and heparan sulphate during virus infection.
Results: A clear correlation was demonstrated between integrin expression and sites of FMDV replication. This work established integrin avb6 as the receptor most likely used by FMDV to target epithelial cells in the bovine hosts. Somewhat unexpected results were also obtained, showing that the role of heparan sulphate may not be confined to cell culture adaptation of virus. Heparan sulphate chains were found to be required for correct assembly of the integrin avb6. In addition, avb6 receptors lacking heparan sulphate chains showed a reduced specificity for RGD containing ligands typical of the cell binding receptor in the VP1 G-H loop of FMDV.
The studies have identified the importance of integrins, in particular integrin avb6, for FMDV infection. Moreover, it is now known that heparan sulphate chains provide essential requirements for correct integrin assembly at the surface of cells. This is of particular importance for vaccine manufacturers, due to the fact that cell culture passage of FMDV creates virus with high heparan sulphate binding capacity. These studies may be also prove useful for the design of antiviral substances specifically inhibiting virus binding to target epithelial cells.
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