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Journal of Virology, May 2005, p. 6410-6418, Vol. 79, No. 10
0022-538X/05/$08.00+0 doi:10.1128/JVI.79.10.6410-6418.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 ONF, England
Received 2 September 2004/ Accepted 6 December 2004
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ABSTRACT |
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INTRODUCTION |
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Clinical signs of the disease vary in severity between species, with cows and pigs showing greater severity of signs than sheep (6). The most obvious effects of FMDV infection are elevated temperature and the appearance of lesions on the tongue and epidermis adjacent to the hoof, namely, the coronary band and, in ruminants, interdigital skin. Lesions first appear in animals which have been infected by contact with infected animals within 1 to 5 days of contact, depending on the intensity of contact, the virulence of the virus, and the dose used to inoculate the pigs. Inoculated animals have viremia from around 1 day after inoculation, and it is assumed that the spread of the virus within such infected animals is by the general circulation.
The precise nature of the virus-cell interactions that give cell type specificity in vivo is not certain, although host cell integrins have been proposed as receptors for FMDV (11-16, 19-21). Both 
vß3 and vß6 integrins have been proposed as cell membrane receptors for FMDV in vivo, and we have recently demonstrated, with bovine epithelium, that vß6 is expressed at considerably higher levels than vß3 on epithelial cells. The highest levels of vß6 were detected in epithelial cells of the tongue and interdigital skin, which are sites most likely to show lesion formation in FMDV infection of cattle (P. Monaghan et al., unpublished data).
Previous studies of lesion formation have identified areas of increased cytoplasmic eosinophilia within the stratum spinosum of the cornified, squamous epithelium as the first stages of lesion formation. This is followed by fluid accumulation in the affected region to form a vesicle which may burst to give the large lesions characteristic of the disease (3, 6). While histological studies of lesion formation have shown that they occur within the epithelial cell layer, the precise route of infection, the cell types involved, and means of local spread are not clear. There have been attempts to study this using in situ hybridization methods (9, 10), but such methods have so far not had a sufficient morphological resolution and do not separate viral replication and accumulation. This is best achieved using immunolabeling and confocal microscopy of viral replication proteins. However, it has been exceedingly difficult to detect viral proteins resulting from viral replication within infected tissues despite many attempts. Away from the main lesion, small microscopic areas where the cells showed cytoplasmic changes have been proposed as early signs of viral replication, but without overt evidence of viral replication, these studies remain speculative (3, 6). Clearly, identifying sites of viral replication in the context of lesion formation and, particularly, individual infected cells will be important in developing our understanding of the early processes of virus replication and pathogenesis of FMDV in vivo.
We report the results of a combined confocal microscopic and reverse transcription-PCR (RT-PCR) study of foot-and-mouth disease in infected pig epithelium. Lesion formation was studied by immunofluorescence labeling for the presence of the nonstructural protein 3ABC in epithelial tissues from FMDV-infected pigs. The 3ABC protein, or rather its precursor P3 in processed form as 3A, 3BCD, 3AB, and 3CD, is expressed at high levels in infected cells, and reaction of intracellular antigens with antibodies raised against FMDV 3ABC protein is indicative of viral replication. The methods used have the sensitivity to detect viral proteins in infected cells and also have the advantage of retaining the morphology of the tissue to allow a full understanding of the processes taking place in infected epithelia. Samples of tissue adjacent to those used for microscopy were processed for RT-PCR to determine the copy numbers of FMDV RNA within the tissue, and these provide a comparative overall picture of virus replication in the tissues studied.
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MATERIALS AND METHODS |
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Animals. The 13 pigs described in this report were from a larger experiment involving 40 Landrace cross-bred Large White pigs weighing between 20 and 30 kg and housed in biosecure animal buildings. Details of the larger experiment will be described elsewhere (C. Murphy, M. Quan, and S. Alexandersen, unpublished data). Briefly, four of the pigs were sacrificed as normal controls, and the other 36 pigs were randomly divided into six separate groups of 6 pigs, of which 3 pigs in each group were inoculated and the other 3 pigs kept as direct contacts. Two inoculated and two direct-contact pigs were selected at random and sacrificed at predetermined time points. The pigs described in this report included one control pig and two inoculated pigs sacrificed at time points 6 h and 1, 2, 3, 4, and 5 days post inoculation (p.i.), as shown in Table 1. Pigs were inoculated in the heel pad as described previously (1, 2). This method of infection, rather than using contact with an infected animal(s), was chosen for this experiment as it allows the time postinfection to be unambiguously known. The animals were examined clinically for signs of FMD, and rectal temperatures were recorded daily.
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For RT-PCR, tissue samples were collected postmortem from pigs randomly selected and sacrificed at the indicated time after inoculation and were put immediately into RNAlater (Ambion, United Kingdom) and then stored at –20°C until required (results to be described in detail elsewhere). For microscopic analysis, samples of the heel pad inoculation site were obtained at 6 h p.i. and samples of tongue, soft palate, coronary band, interdigital skin, and flank skin were obtained at days 0 (control), 1, 2, 3, 4, and 5 p.i. (Table 1). Where a lesion could be detected macroscopically, the sample was taken to include both lesion and surrounding apparently normal skin. The samples, approximately 1 to 2 cm by 0.5 to 1.0 cm, were immediately placed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 90 to 120 min and then transferred to PBS for storage at 4°C.
For sectioning, the method was essentially as described previously (17, 18). The tissues were trimmed to an oblong shape if needed and then attached to the holder of a vibrating microtome (Leica Microsystems, Milton Keynes, United Kingdom) with superglue. They were sectioned and processed under PBS. The short fixation time used in this study provides the optimum combination of minimal damage to antigens with preservation of tissue structure. Sections (70 µm) were cut as near to 90° to the surface of the tissue as possible. The sections were handled with paint brushes trimmed to one bristle and immunolabeled in 150-µl droplets of reagent. The sections were permeabilized for 60 min in 0.1% Triton X-100, nonspecific labeling was blocked with PBS containing 0.5% bovine serum albumin (PBS-BSA) overnight, and then the sections were transferred to primary antibody for 90 min. Labeling for FMDV replication was with a rabbit antibody recognizing the nonstructural protein 3ABC (IAH) diluted 1:1,000 in PBS-BSA. Following 10 2-min washes, the bound primary antibody was detected with species-specific antibodies conjugated to Alexa 488 (Molecular Probes, Cambridge Biosciences, Cambridge, United Kingdom) diluted 1:200 in PBS-BSA for 90 min. Ten 2-min washes were followed by 30 min in DAPI (4',6'-diamidino-2-phenylindole) (Sigma, Poole, United Kingdom) diluted 1:5,000 in PBS and mounting in Vectashield (Vector Laboratories, Peterborough, United Kingdom). Coverslips were sealed with nail varnish.
Control sections were labeled in the absence of primary antibody or with an irrelevant primary antibody. The sections were imaged in a Leica SP2 confocal microscope equipped with 405-, 488-, and 568-nm laser excitation (Leica Microsystems, United Kingdom). Differential interference contrast (DIC) images were also collected.
Quantitative RT-PCR. Although the vibrating microtome sections provide relatively large pieces of tissue for analysis, there remains a risk of sampling error. In order to make a correlation between immunolabeling and overall viral replication, we determined the quantity of FMDV RNA in extracts of total nucleic acid from blood and tissue samples by quantitative real-time RT-PCR. The assay method detects FMDV RNA in the 5' untranslated region and has been described in detail elsewhere (5, 7, 26-28). All extractions involved 0.200 ml of sample, the nucleic acids were finally eluted in a volume of 0.050 ml, and all estimations included standard reactions based on in vitro-transcribed, molecularly cloned FMDV RNA, using samples with a known content of FMDV genome equivalents (31). The method is influenced minimally by sample type (4-6, 22, 26, 28, 29).
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RESULTS |
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Briefly, an increase in body temperature, viremia, and early local lesions were detected in the inoculated pigs from around 24 h after inoculation and reached a peak at 48 h after inoculation with severe, generalized lesions. Clinical disease was severe and characterized by lameness/reluctance to stand or walk, reduced or no interest in feed, and severe vesicular lesions along the coronary bands and heel pad area and sometimes on the tongue and snout. The details for the pigs examined here will be given together with the microscopic findings below.
Microscopic findings. No vesicular lesions were observed in the soft palate, interdigital skin, and flank skin, and no labeling was detected in samples from these regions at all stages of the experiment. The viral load in the soft palate samples as measured by quantitative RT-PCR is reported below. The viral loads in interdigital skin and flank skin were not measured for these samples; however, previous experiments indicate that the viral loads in these tissues are in general only 0.1 to 1% of the levels in skin with vesicular lesions and never exceed 108 FMDV copies per gram of tissue (S. Alexandersen, M. Quan, and C. Murphy, unpublished findings).
Day 0 (control), 6 h, and day 1. Macroscopically, no lesions were observed in the control pig. In the pigs at 6 h p.i. the inoculation sites were visible on the heel pads as needle tracks but without any vesicular lesions. In the pigs at 1 day p.i. (VA85 and VA86) local vesicular lesions were observed at the sites of inoculation on the heel pads, but no generalized lesions were evident and their body temperatures were not elevated (38.6°C and 38.9°C, respectively). No labeling was detected by confocal immunofluorescence microscopy at any location in tissues from the control animal or animals at 6 h or 1 day p.i. This does not prove that there were no infected cells within the coronary band or tongue, just that the lack of lesions made the selection of the site of the epithelium for microscopy an arbitrary process.
Examination of coronary band epithelia, tongue, and soft palate for FMDV RNA showed that all the day 0 samples and the 6-h-p.i. samples were negative (below the detection limit of around 104.2 copies per gram), while the 1-day-p.i. samples contained from 105.4 to 108 copies of FMDV RNA per gram of tissue, except for the inoculated heel pad tissue from pig VA85, which contained around 1010.7 copies of FMDV RNA per gram. Both VA85 and VA86 had viremia of around 108 copies of FMDV RNA per ml (Table 1).
Day 2. Macroscopically, the pigs at 2 days p.i. (VA79 and VA80) had generalized lesions involving all four feet and the tongue, and their body temperatures were elevated to above 40°C.
Lesions of the tongue were well developed, and samples were taken which contained both lesion and adjacent apparently uninvolved tissue. In the tongue, the epithelium contains many cell layers, from the basement membrane through the spinous layer to the upper cornified layer, which has specialized "hairs" on the surface. The basal region of the epithelium has deep invaginations (papillae), which are long processes of the dermis consisting of capillaries and stromal cells and extend almost to the outer cornified layer of the epithelium. Immunofluorescence labeling of the edge of the lesion showed that FMDV 3ABC protein was present in most layers of the tongue epithelium, extending from the basal area up to close to the cornified layer (Fig. 1a and b). Large numbers of infected cells were present in the region of lesion, but the infected cells were detected only within the cells of the epithelium. No positively labeled stromal cells were seen. The lesion on the tongue consisted of a split within the epithelial layer which had occurred some way up away from the basal level in the region of the stratum spinosum. A small number of FMDV-positive cells remained below the lesion. This is more clearly seen in Fig. 1c and d, which show the center of a lesion. The cells of the epithelium separated from the dermis (upper region in Fig. 1) are almost all FMDV positive, and below the lesion, clusters of FMDV-positive cells are also seen in the epithelium remaining in contact with the dermis. The tissue architecture in this region was distorted, and from this time postinfection onward, the whole area of the lesion along with its associated dermal tissue was infiltrated with a massive influx of small cells with a high nuclear/cytoplasmic ratio. These were interpreted as being lymphocytic cells.
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While there were areas of infection where the basal cells were clearly labeled (Fig. 2a and b) in the majority of lesions observed, the basal cells were not, at least at this stage, infected (Fig. 2c). Whether any or all of these smaller lesions were interlinked is unclear, as the papillae gave the impression of interrupting the infected area. Our interpretation of the images is that it is likely that the infected areas formed interlinked lesions rather than a group of separate lesions.
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Examination of coronary band epithelia, tongue, and soft palate for FMDV RNA showed that the 2-day-p.i. samples (VA79 and VA80) contained from around 108.3 copies of FMDV RNA per gram for the soft palate tissue up to 1010.7 to 1011.5 copies per gram for the tongue, coronary band, and heel pad. Both VA 79 and VA80 had viremia of around 109.5 to 109.8 copies of FMDV RNA per ml (Table 1).
Day 3. Macroscopically, the pigs at 3 days p.i. (VA73 and VA74) had severe generalized and ruptured lesions involving all four feet and tongue, and their body temperatures were elevated to above 40°C. By confocal immunofluorescence microscopy, the labeling intensity in the region of the lesion in the tongue was dramatically reduced compared to that for the day-2-p.i. animals, and positive cells were only rarely detected at the edge of the lesion. There was a clear dividing line at the edge of the lesion between the necrotic tissue of the lesion and the apparently normal residual epithelium (Fig. 3a and b).
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Examination of coronary band epithelia, tongue, and soft palate for FMDV RNA showed that the 3-day-p.i. samples (VA73 and VA74) contained around 106.7 copies of FMDV RNA per gram for the soft palate tissue, while the tongue samples contained from 107.2 (VA74) to 108.6 (VA73) copies per gram and the coronary epithelia up to 109.4 (VA74) and 1010.4 (VA73) copies per gram (Table 1). VA73 had viremia of around 108.1 and VA74 of around 107.3 copies of FMDV RNA per ml.
Day 4. Macroscopically, the pigs at 4 days p.i. (VA67 and VA69) had severe generalized and ruptured lesions involving all four feet and tongue, and their body temperatures, which had been 40.0 and 39.8°C on days 2 and 3 p.i., had dropped to 39.6 and 39.3°C, respectively.
In the region of the tongue lesion there was no clear labeling for FMDV by confocal immunofluorescence microscopy. In the coronary band lesion, there were still positively labeled cells within the lesion. (Fig. 3e).
Examination of coronary band epithelia, tongue, and soft palate for FMDV RNA showed that the 4-day-p.i. samples (VA67 and VA69) contained from around 106.4 to 106.8 copies of FMDV RNA per gram for the soft palate tissue, while the tongue samples contained from 107.0 (VA67) to 108.3 (VA69) copies per gram and the coronary epithelia up to 109.0 copies per gram (Table 1). VA67 had viremia of around 103.7 and VA69 of around 103.9 copies of FMDV RNA per ml.
Day 5. Macroscopically, the pigs at 5 days p.i. had different severities of lesions. Pig VA81 had severe generalized and ruptured lesions involving all four feet and tongue and a body temperature of 39.6°C, while pig VA87 had lesions only on the inoculated foot and on the tongue and had a body temperature of 39.5°C.
By confocal immunofluorescence microscopy examination, the lesion on the tongue of pig VA81 showed no positive labeling. The lesions on the coronary bands of both animals showed an overall picture of complete loss of epithelial architecture with mostly necrotic epithelium. However, there was still extensive labeling in the lesion, and this was clearly associated with individual cells (Fig. 3f). Both tongue and coronary band lesions contained a large number of small cells with round nuclei that were considered to be lymphocytes. In the tongue lesion, there was, however, a hint of a return of a papillary structure, which may indicate the emergence of a few uninfected basal epithelial cells from within the stroma below the lesion.
Examination of coronary band epithelia, tongue, and soft palate for FMDV RNA showed that the 5-day-p.i. samples (VA81 and VA87) contained from around 104.8 copies of FMDV RNA per gram for the soft palate tissue, while the tongue samples contained from 106.4 (VA81) to 107.0 (VA87) copies per gram and the coronary epithelia contained 108.3 (VA87) and 1010 (VA81) copies per gram (Table 1). Neither VA81 nor VA87 had detectable viremia (detection limit around of around 102.4 copies of FMDV RNA per ml).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generalized skin lesion formation begins after the animals become viremic and it must be assumed that the susceptible cells in the cornified squamous epithelia become infected by the virus traversing the basement membrane. The mechanism for this is not known, and it is not clear if this is an active or passive process (6). Lesion formation takes place rapidly within infected animals, but this is not too surprising as it has been reported that the cytopathic effect of FMDV can be detected in BHK-38 cells, which are a particularly susceptible tissue culture cell line, as soon as 1.5 h p.i. (17). While this is an extreme case, the doubling rate of FMDV in pigs has been estimated to around 2.3 h (Quan and Alexandersen, unpublished). Extrapolation of data from tissue culture to the in vivo situation is clearly an uncertain exercise and supports the need for experimental information as described here.
The targeting of FMDV exclusively to the epithelial cells in the tongue and coronary band epithelium is clear from this study; however, the mechanism is not clear. The outer capsid of FMDV consists of 60 copies of each of four proteins, VP1, VP2, VP3, and VP4. The external loop of VP1 has a highly conserved RGD motif, and this is a well-recognized ligand for the class of cell adhesion and signaling molecules known as integrins (11-16, 19-21). These are complex molecules which consist of heterodimers of two integrin families, and ß, and both vß6 and vß3 have been proposed as receptors for FMDV, thus providing a possible mechanism for the cell type specificity observed. Indeed, tissue culture studies have indicated that FMDV binds to and is internalized into susceptible cells by means of these cell surface integrin molecules (12-16). The pattern of expression of integrin molecules vß6 and vß3 observed within the epithelium of bovine tissues is in line with the results reported here (Monaghan et al., unpublished data), but further work will be needed to determine if the integrin expression pattern of porcine epithelium is similar to that seen in bovines.
Our interpretation of the data presented here suggests that the first site of infection in the epithelium is either a basal epithelial cell on the basement membrane or an epithelial cell further "up" the epithelium within the stratum spinosum. Certainly in infected regions away from an active lesion, the infected cells do not always include the basal cells, and this location of infection above the basement membrane correlates well with the formation of the lesion some way above the lower limit of the basement membrane. This lesion formation above the basement membrane left patches of epithelial cells below the lesion. While many of these cells were positively labeled, it was not possible to determine if all the epithelial cells became infected. The apparent beginnings of restoration of tissue structure in the day 5 p.i. tongue sample might suggest that a small number of epithelial cells escape infection and act as a source of new epithelial cells or, alternatively, that the FMDV infection may not necessarily be cytolytic in basal cells.
Away from the lesion, the infected cells were seen around the epithelial papillae, and at no stage was any cell seen outside the basement membrane which labeled positive for the presence of 3ABC. This may indicate that the spread of the virus within the epithelium is predominantly from epithelial cell to cell, and this may be encouraged by cellular stress or trauma (6).
The results of the microscopic examination and the RT-PCR are in agreement on the rapid decline in FMDV replication in the tongue compared with the coronary band. On day 2, when both tongue and coronary band show high levels of 3ABC expression, the RT-PCR gives values of 1010.7 to 1011.5 viral genomes per gram. In contrast, on day 3 p.i., the microscopy of the coronary band showed strong labeling, with almost no labeling present in the tongue lesion. The RT-PCR of coronary band showed similar levels to the day 2 samples (109.4 to 1010.4), whereas the tongue levels were 107.2 to 108.6. Why the viral replication appears to decline more slowly in coronary band lesions is not clear. However, one possibility is the influence of the host response to the infection. The edge of the lesion in the tongue where it abuts the remaining normal epithelium in particular was very distinct, and it may be that the onset of host defenses limits lesion growth by inhibition of viral replication and that these host responses are more acute in the oral region. Certainly the lymphatic system is well developed in this region compared to epithelium in the hoof region. In addition, there is some evidence that FMDV antigen and genome are maintained longer in foot lesions than in tongue lesions, which may be due to various mechanisms, e.g., a prolonged period of active replication, increased stability, decreased clearance, or simply differences in cell renewal rate (6, 23-25, 30).
The soft palate has been proposed as a possible route of entry of the virus, and the failure to detect viral replication in the soft palate may have a number of causes. While it may be a matter of detection sensitivity, in that no cells expressed sufficient 3ABC to be detected, it may be that infection is patchy and the sections prepared for confocal microscopy did not contain infected cells. Comparison of the RT-PCR data and the immunolabeling results as a whole does indicate that the microscopic detection limit is around 108 copies per g of tissue, although coronary band tissue of pig VA67 was positive by immunolabeling and contained only 105.3 genomes per gram. This may be due to a focal distribution of infected cells. As already discussed, these two systems detect different aspects of viral replication, but on this basis, the levels in the soft palate were not significantly above the detection limit. Another possibility is that the RNA detected by RT-PCR could result from passive uptake of virus within the tissue or, alternatively come from a cell type other than the epithelium itself, such as mucus glands or nasal-associated lymphoid tissue.
In all cases with lesion formation there was a massive lymphocytic infiltrate throughout the lesion both above and below the basement membrane, and further studies will follow this process in more detail.
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ACKNOWLEDGMENTS |
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This research was supported by the Department for Environment, Food and Rural Affairs (DEFRA), United Kingdom; the Biotechnology and Biological Sciences Research Council (BBSRC), United Kingdom; and the EU (project number QLK2-CT-2002-01719).
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß1 is a receptor for foot-and-mouth disease virus. J. Virol. 76:935-941.vß6 is a receptor for foot-and-mouth disease virus. J. Virol. 74:4949-4956.
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