Quantitative analysis of foot-and-mouth disease virus RNA loads in bovine tissues: implications for the site of viral persistence

Zhidong Zhang and Soren Alexandersen

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK

Correspondence
Soren Alexandersen
soren.alexandersen{at}bbsrc.ac.uk


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To understand better the pathogenesis of foot-and-mouth disease (FMD), the levels of viral RNA in various bovine tissues during the acute and persistent stages of FMD virus (FMDV) infection were investigated by using quantitative RT-PCR. The viral RNA levels in the tissues examined had peaked by day 1 post-infection (p.i.) and were markedly different among the tissues examined. The epithelium collected from sites of lesion development, i.e. the interdigital area and coronary band on the feet, and the tongue, contained the highest level of viral RNA, indicating the predominant tissue sites of viral infection and amplification during the acute stage of infection. Clearance of viral RNA from most of the tissues occurred relatively rapidly and the rate of clearance was largely independent of the level of viral RNA. The viral RNA load in most of the tissues declined slower than in serum, in which viral clearance is rapid. Beyond 28 days p.i., a proportion of pharyngeal region tissues (soft palate, pharynx, tonsil and mandibular lymph node) from infected animals still contained a detectable level of viral RNA, while viral RNA in non-pharyngeal region tissues was generally only detectable for variable periods ranging from 4 to 14 days p.i. The presence of viral RNA in dorsal soft palate tissue had a good correlation with the presence of infectious virus in oesophageal-pharyngeal fluid (OP-fluid) samples, a finding indicative of the specific tissue sites of FMDV persistence.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae, genus Aphthovirus that causes a highly contagious disease in cloven-hoofed animals characterized by the appearance of vesicles on the feet and in and around the mouth (Alexandersen et al., 2003b; Bachrach, 1968). Usually the mortality in adult animals is negligible but it may be considerable in young animals. The severe impairment of production capabilities and the slaughter of affected animals to eradicate the virus following outbreaks can result in enormous economic losses. The 2001 outbreak of foot-and-mouth disease (FMD) in the UK and mainland Europe is a timely reminder of its devastating consequences. Apart from causing acute infection and disease, FMDV is able to cause persistent infection (so-called carriers) in ruminants, which may also occur in vaccinated ruminants exposed to live virus (Burrows, 1966; Rossi et al., 1988; Straver et al., 1970; reviewed recently by Alexandersen et al., 2002b). Such carriers may be critical to the epidemiology of FMD (Dawe et al., 1994; Hedger & Condy, 1985) and make control efforts even more costly. A carrier is defined as an animal from which live virus can be recovered for longer than 28 days after exposure (Sutmoller & Gaggero, 1965; Salt, 1993; Alexandersen et al., 2002b). During the period of persistence, there is considerable variation in the levels of virus recovery from oesophageal-pharyngeal fluid (OP-fluid) samples. Persistent infection appears to occur in a variable proportion of virus-exposed ruminants and eventually become undetectable in the carriers (Alexandersen et al., 2002b).

A number of studies have shown the importance of the pharyngeal area tissues in FMDV infection during acute disease and persistence (Burrows et al., 1971; McVicar & Sutmoller, 1969; Prato Murphy et al., 1994; Zhang & Kitching, 2001). Bovine pharyngeal tissue has been identified as a primary site for FMDV infection in vivo (Burrows et al., 1971; McVicar & Sutmoller, 1969). The sites of FMDV replication in persistently infected cattle have been examined by the recovery of infectious virus from OP-fluid samples (Burrows, 1966), and by using RT-PCR to detect viral RNA in bovine pharyngeal epithelia samples (Prato Murphy et al., 1994) of carrier animals. Although viral RNA was recently localized in epithelial cells of the bovine pharyngeal area (Zhang & Kitching, 2001), the source of the virus in vivo remains elusive and thus the molecular mechanisms of FMDV persistence remains to be fully understood. The duration of persistence of the virus might be affected by the rates of residual viral RNA replication, degradation/clearance of viral RNA or by the particular type of cells infected. The quantitative analysis of viral RNA loads can shed light on the effect of these mechanisms on the fate of the virus. Therefore, viral RNA levels in bovine tissues during the acute and persistent stage of FMDV infection were investigated by using a quantitative RT-PCR assay described previously (Alexandersen et al., 2001, 2002c; Oleksiewicz et al., 2001; Reid et al., 2001, 2002, 2003). Viral RNA levels in the various tissues were quantified over a 72-day period following initiation of infection. This study yielded valuable insight into the viral RNA kinetics in different tissues during acute disease and persistence.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Experimental animals and samples.
Thirty standard Compton steers (Holsteins) at 6–10 months of age were used. This group included four uninfected animals used as controls. Uninfected-control animals were slaughtered and tissue samples were collected as for the infected animals (see below). Animals for the infection studies were placed as pairs of two or four together in individual rooms in a biosecure animal building. Inoculation was by subepidermo-lingual injection of approximately 0·5 ml (6·9 log10 TCID50) of FMDV O UKG 34/2001 as described elsewhere (Alexandersen et al., 2002a, 2003a; Alexandersen & Donaldson, 2002; Zhang et al., 2004). For experiments that included contact infection, half of the animals in each room were selected at random and inoculated, while the other half of the animals were kept as direct-contact animals throughout the experiment. After inoculation, animals were monitored for clinical signs of disease and the rectal temperatures were recorded daily until 10 days after inoculation. Samples of OP-fluid and blood (heparinized and non-stabilized for serum) were collected before the start of the experiment (negative controls) and at intervals after infection as described previously (Zhang et al., 2004) and are indicated in Table 1. OP-fluid samples were collected using a standard cattle probang cup (Sutmoller & Gaggero, 1965) and diluted with an equal volume of Eagle's-HEPES medium (pH 7·2) containing 5 % fetal calf serum (FCS), and stored at –80 °C until required.


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Table 1. Clinical profiles and recovery of FMDV in the OP-fluid samples of experimentally-infected animals

 
To isolate peripheral blood mononuclear cells (PBMC), heparinized blood was centrifuged, and the buffy coat was resuspended in Ca2+/Mg2+-free PBS, underlayed with Histopaque-1077 (Sigma) and centrifuged at 600 g for 30 min. Cells at the interface were collected and washed (600 g for 5 min) in Ca2+/Mg2+-free PBS. Cell pellets were stored at –80 °C until use.

Tissue samples (Table 2) were collected at post-mortem from cattle randomly selected and killed at the indicated time after infection and after selection the samples were immediately put into RNAlater (Ambion), then stored at –20 °C until required. Similar tissue samples were also available from cattle included in previous experiments including: four cattle infected with FMDV O BFS 1860 and five cattle infected with FMDV O SKR 1/2000, as well as two cattle infected with FMDV C Oberbayern (Zhang & Kitching, 2000; Zhang et al., 2004). The results obtained from these cattle samples will only be mentioned briefly.


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Table 2. Kinetics of FMDV RNA loads in bovine tissues during acute infection

Tissue samples were collected from cattle experimentally infected with FMDV O UKG 34/2001 as described in Methods. Four uninfected cattle were killed as controls and tissues collected for analysis. ND, No samples taken.

 
Assay for virus.
Each OP-fluid sample was assayed in primary bovine thyroid (BTY) cells to determine the presence of infectious virus as described (Snowdon, 1966).

RNA extraction.
Briefly, for each approximately 20 mg tissue sample to be processed, 600 µl MagNA Pure LC mRNA isolation kit II (Tissues) lysis buffer (Roche) was added to a tube containing Lysing Matrix D (Bio 101 Systems; Qbiogene). The tube was processed in the FastPrep centrifuge (Bio 101) for 45 s and incubated on ice for 1 min (this step was repeated twice) according to the manufacturer's instruction. After being centrifuged at 8000 g in a Biofuge (Heraeus) centrifuge for 5 min at 4 °C, 300 µl tissue lysate was carefully transferred to a new microcentrifuge tube avoiding to transfer the pelleted debris and lysing matrix. RNA was then extracted by using MagNA Pure LC mRNA extraction kit II (Tissues) with an automated nucleic acid robotic workstation (Roche) according to the manufacturer's instruction. RNA was eluted in 50 µl of elution buffer (Roche) and stored at –80 °C until used. Up to 105 PBMCs were used for extraction of RNA by using MagNA Pure LC mRNA extraction kit I (Roche). For serum and OP-fluid samples, 200 µl samples were mixed with 300 µl MagNA Pure LC total nucleic acid lysis buffer (Roche). Total nucleic acids were extracted and eluted in 50 µl elution buffer by using MagNA Pure LC total nucleic acid isolation kit (Roche) as described previously (Alexandersen et al., 2003a). RNA from tissue samples collected from cattle infected with FMDV O SKR 1/2000, O BFS1860 and C Oberbayern was extracted by using 1 ml of TRIzol (BRL) per 100 mg of sample according to the manufacturer's protocol.

Quantitative RT-PCR assay for detection of viral RNA.
The level of viral RNA in samples was quantified by a quantitative RT-PCR assay as described previously (Alexandersen et al., 2002c, 2003a; Reid et al., 2002, 2003; Zhang & Alexandersen, 2003). In vitro transcribed RNA used to generate standard curves was synthesized as described previously (Zhang et al., 2002, 2004).

Calculation of the rates of viral RNA clearance/decay.
Rates of viral decay or clearance were calculated by using the decay rate equation ({alpha})=(lnY1–lnY2)/(t1–t2), where Y1 and Y2 are the virus load at times t1 and t2, respectively (Zhang et al., 2004). The viral decay/clearance half-life, T1/2 in h, was calculated by using the equation T1/2=(ln2)/{alpha}. Viral decay rates and T1/2 were calculated by using data points between day 1 and 6 p.i.

Statistical analyses.
Pearson's coefficient of correlation (r) and the statistical significance of r were tested using MINITAB release 12.21 software. A P value <0·05 was considered statistically significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Clinical profiles of cattle experimentally infected with FMDV
All animals inoculated with FMDV O UKG 34/2001 developed signs of clinical disease within 1–2 days p.i. as described previously (Zhang et al., 2004). One animal died unexpectedly and was excluded from this study. An increase in body temperature, viraemia and early lesions were observed in all inoculated cattle from around 1 day p.i. and in the contact cattle at around 3–5 days p.i. Vesicular lesions were found on the tongue of inoculated cattle from around 1 day p.i. and in the interdigital area or along the coronary band on feet from around 2 days p.i. Lesions in contact animals were detected at 3–5 days p.i. on the tongue, dental pad and lips and in the interdigital area or along the coronary band on two or more feet. Viral RNA and infectious virus in OP-fluid samples were positive by a quantitative RT-PCR assay and virus isolation assay in BTY cells from around 1 day p.i. (data not shown) (Alexandersen et al., 2003a; Zhang et al., 2004). Cattle were confirmed as carriers of FMDV by recovery of infectious FMDV from OP-fluid samples collected at 28 days p.i. (Table 1). Results from cattle infected with FMDV O BFS 1860 and C Oberbayern were essentially similar, while cattle inoculated with FMDV O SKR 1/2000 showed only very mild local lesions without generalization (no lesions at other sites apart from tongue) and were negative for infectious FMDV in OP-fluid samples at 11 days p.i. and later (data not shown).

Kinetics of viral RNA levels in bovine tissues during the acute stage of infection
The kinetics of FMDV infection in bovine tissues during acute infection were studied by quantifying viral RNA levels in a panel of tissue samples collected at 0, 1, 3, 4, 5, 6 and 14 days p.i. from the animals experimentally infected with FMDV O UKG 34/2001 described in Table 1. Viral RNA levels in serum and OP-fluid samples were also quantified in order to identify the viraemic phase or viral peak level in OP-fluid samples during acute infection. The data are summarized in Table 2. The viral RNA levels in the tissues examined peaked on day 1 p.i. and were markedly different in the various tissues. The peak level of viral RNA in tissues was at a lower or similar level as in the serum and OP-fluid samples except for the tongue lesion samples that were higher. The level of viral RNA in tissues at 1 day p.i. varied from 6·23 to 11·46 log10 copies g–1 tissue. Among the tissues examined from these animals, the epithelium collected from interdigital area and coronary band on the feet (EPI) and tongue samples contained the highest level of viral RNA. After peak levels, FMDV RNA levels in tissues decreased by various rates but were still high at 3–4 days p.i. (Table 2). At day 6 p.i., a marked reduction in RNA copy number occurred. Although, there was no detectable viraemia at this time most of the tissues examined, except heart tissue, still contained a quantifiable level of viral RNA with a maximum level of 8·02 log10 copies g–1 tissue in EPI with lesions (Table 2). A similar distribution of FMDV RNA was also observed in the tissues collected at day 3 p.i. from animals infected with FMDV O BFS 1860 and O SKR 1/2000, although the levels for O SKR 1/2000 was relatively low (data not shown). At 14 days p.i. [only one animal (UU74) available as one animal died early], most of the tissues examined were negative for viral RNA except tongue and tonsil, which contained a detectable level of viral RNA. Virus isolation from an OP-fluid sample of this animal was negative [isolation of virus-negative (IV-negative)].

Viral RNA clearance/decay in tissues during the acute stage of infection
In exploring the reduction in RNA levels between 1 and 6 days p.i., viral RNA clearance/decay rates in tissues during the acute stage of infection were calculated and are summarized in Table 3. Viral RNA clearance/decay rates in both dorsal soft palate (DSP) and nasopharynx were slower than in the other tissues including ventral soft palate (VSP), oropharynx and tonsil. Clearance/decay in retropharyngeal lymph node (RPLN) and cervical superficial lymph node (CLN) was similar to these latter tissues while clearance/decay in mandibular lymph node (MLN) was particularly slow, possibly because of drainage of virus/viral RNA from soft palate and pharynx. The viral RNA load in serum declined faster (corresponding to a half-life of 4·6 h calculated as described previously; Zhang et al. 2004) when compared with tissues, and became undetectable by day 5 p.i. coinciding with the antiviral antibody response (data not shown). Thus, antiviral antibody effectively cleared virus in serum, while clearance of viral RNA from tissues, in particular DSP, nasopharynx and MLN, was significantly delayed.


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Table 3. Viral RNA clearance/decay in tissues during acute stage of infection

 
Persistence of viral RNA in tissues
The pharyngeal area tissues have been shown to be important in FMDV infection during both acute infection and persistence (Alexandersen et al., 2003b). Therefore, the duration of FMDV infection was investigated in tissues collected after 28 days p.i. from the pharyngeal region including DSP, VSP, nasopharynx and oropharynx, tonsil, MLN and RPLN as well as from other non-pharyngeal tissues from FMDV O UKG 34/2001-infected cattle (Table 1). No OP-fluid sample was taken from animal UO46 at the time of sacrifice and data from this animal were not included for analysis. Six of fourteen animals examined were IV-positive in OP-fluid samples and eight of them IV-negative at the time when the animals were killed. Data summarized in Table 4 show that viral RNA persisted in certain pharyngeal region tissues at detectable levels beyond 28 days p.i. from some infected animals. Among these tissues, DSP tissue from all six animals with OP-fluid samples positive for virus at the time of sacrifice contained a detectable level of viral RNA, ranging from 4·22 to 6·28 log10 copies g–1 tissues. Among these six animals with OP-fluid samples positive for virus, three of the VSP, nasopharynx, oropharynx and MLN contained viral RNA at a level around 3–5 log10 copies g–1 tissue, while a single CLN sample contained a low level of viral RNA. Among the eight animals with OP-fluid samples negative for virus, only one DSP, two VSP and one nasopharynx samples contained viral RNA at levels from around 3–5 log10 copies g–1 tissue. There was no viral RNA detected in tissues of tongue, EPI, MLN, CLN, tonsil, lung, heart, liver and spleen collected after 28 days p.i. from any of these animals (Table 4). Similar patterns were also observed in tissues from carrier FMDV C Oberbayern- and O BFS 1860-infected animals with detectable levels of viral RNA only consistently detected in the pharyngeal region (data not shown). The results suggested that non-pharyngeal sites of viral RNA persistence are most unlikely to serve as a significant tissue site for FMDV infection and replication during persistence.


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Table 4. Persistence of FMDV RNA in bovine tissues

Tissue (n=14) samples were collected after 28 days p.i. from experimentally-infected cattle with FMDV O UKG 34/2001 as described in Methods. ND, This sample was not tested by real-time RT-PCR.

 
The rate of clearance in tissues is independent of the initial levels of viral RNA
To correlate viral RNA load with the duration of virus persistence in tissues we chose to analyse viral RNA levels at day 6 p.i., because at this time no RNA was detectable in serum, thus ensuring that viral RNA levels present in the tissues studied reflected virus replication or accumulation rather than a result of viral RNA in blood. The highest levels of viral RNA were observed in the tongue and EPI, with intermediate levels in the soft palate, nasopharynx, tonsil, MLN, CLN and RPLN, and lower levels in the oropharynx, lung and spleen (Table 2). Correlation of viral load with the duration of virus persistence in tissues was then analysed. A low correlation between mean viral RNA levels during acute stage of the disease and duration of viral RNA persistence (r=0·07, P=0·8; Pearson's coefficient of correlation) was observed in the tissues, indicating that there is no statistically significant correlation between viral load and the duration of virus persistence in tissues.

PBMCs do not serve as a cellular site for FMDV infection and replication during persistence
Based on dot blot analysis, it has been suggested that FMDV RNA may be associated with PBMCs even during persistent infection (Bergmann et al., 1996). However, whether PBMCs are a reservoir for FMDV infection remains inconclusive. To address this, PBMCs were isolated from FMDV-infected animals and viral RNA was detected by using a real-time RT-PCR assay. In PBMCs isolated from seven FMDV UKG 34/2001-infected cattle (cattle numbered UU in Table 1), only one of seven infected cattle contained a detectable level of FMDV RNA (3·39 log10 copies per 106 PBMCs) at day 2 p.i. and only two of seven infected cattle (2·89±0·74 log10 copies per 106 PBMCs) at day 3 p.i., i.e. at the height of viraemia, and then remained undetectable until the experiment was terminated. In a different experiment on two UKG 34/2001-infected cattle, viral RNA was only detected in PBMCs at day 1 p.i. (2·98±0·14 log10 copies per 106 PBMCs; n=2) and day 2 p.i. (2·76 log10 copies per 106 PBMCs, n=1). There was no viral RNA detected in PBMCs collected at 3–4 days p.i. when these two animals were killed. PBMCs from FMDV O BFS 1860-infected cattle were continually negative when tested after 7 days p.i. until 30 days p.i. (data not shown). Therefore, we conclude from this that PBMCs do not serve as a cellular site for FMDV infection and replication during persistence and either play a minor or no role during acute infection in cattle.

Correlation between the presence of viral RNA in pharyngeal tissues and the recovery of infectious virus in OP-fluid samples
During FMDV infection in cattle, the infectious virus can be isolated from OP-fluid samples of carrier cattle (reviewed by Alexandersen et al., 2002b, 2003b). However, the source of the virus in vivo remains inconclusive, although our previous study has shown that the pharyngeal region is the major site for the virus during persistence (Zhang & Kitching, 2001). As mentioned above, viral RNA persisted in the pharyngeal region at detectable levels after 28 days p.i. in a proportion of infected animals. Therefore, an association of infectious virus detection in OP-fluid samples (by virus isolation in BTY cells) collected after 28 days p.i. with the presence of viral RNA in pharyngeal tissues was studied. The chance of having viral RNA in these pharyngeal region tissues when infectious virus was detected in OP-fluid samples by virus isolation (OP-fluid samples positive for virus) was significantly higher than in tissues from cattle with OP-fluid samples negative for virus (Table 4). There is a substantial agreement (r=0·87, P=0·001) between infectious virus in OP-fluid samples and the presence of FMDV RNA in DSP tissues (n=14) (Table 5). Clearly, viral RNA was detected at a quantifiable level (5·09±0·57 log copy no. g–1 tissues) in six DSP tissues from carriers with OP-fluid samples positive for virus. In contrast, only one (animal UO30) of eight DSP tissues from cattle with OP-fluid samples negative for virus contained a detectable but much lower level of viral RNA (Table 5). The result suggested that DSP tissue might serve as a major source of the virus present in OP-fluid samples. In addition, the chance of having viral RNA in pharynx tissue when infectious virus was detected in OP-fluid samples by virus isolation was also higher than in tissues from cattle with OP-fluid samples negative for virus. Viral RNA was detected at a quantifiable level in three of six nasopharynx tissues from carrier cattle (n=6) with OP-fluid samples positive for virus. In contrast, only one of eight nasopharynx tissues from infected cattle with OP-fluid samples negative for virus contained a detectable level of viral RNA (Table 4). The correlation between infectious virus present in OP-fluid samples and the presence of FMDV RNA in DSP tissue was much higher than in the other pharyngeal tissues (Table 5). There was no relationship between infectious virus in OP-fluid samples and the presence of FMDV RNA in tissues of tonsil and VSP (Table 5). These results were further supported by findings in pharyngeal tissues collected from cattle with FMDV O BFS 1860 and C Oberbayern (data not shown).


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Table 5. Correlation between the presence of viral RNA in pharyngeal tissues and the recovery of infectious virus in OP-fluid samples

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The experiments described here present a quantitative analysis of the levels of FMDV viral RNA in tissues of cattle experimentally infected with FMDV O UKG 34/2001. The predominant sites of FMDV infection were determined to be tissues rich in epithelium such as the tongue and skin of the feet during the acute stage of infection and the pharyngeal region during persistence. The presence of viral RNA in DSP had a good correlation with the presence of infectious virus in OP-fluid samples and is much higher than in the other pharynx tissues.

Although viral RNA was detected in all the tissues examined at 1 day p.i., there were pronounced differences in the levels of viral load among tissues. The load of viral RNA in tongue and EPI was typically 10 to 1000 times greater than the load of FMDV RNA measured in any other tissue. The result of this study reasserts the importance of cornified, stratified squamous epithelia of the tongue and skin as the predominant tissue site of viral amplification. The high level of virus infection and replication in these tissues most likely reflects the abundance of permissive target epithelial cells in these tissues, and therefore plays a key role in the pathogenesis of acute FMD manifested as vesicular lesions on the tongue and feet. Significant levels of viral RNA were observed in pharyngeal tissues collected from all the infected animals during acute disease. However, these levels of viral RNA are relatively low compared with the level measured in tongue and skin, which may indicate low-level virus infection and replication in this region, perhaps because of few target cells or limited (restricted) replication in this target population. These data, together with the previous studies which reported that certain epithelial cells of the pharynx were positive for FMDV RNA (Prato Murphy et al., 1999; Woodbury et al., 1995; Zhang & Kitching, 2000, 2001), imply that pharyngeal tissues are important tissue sites of FMDV infection and replication during both acute infection as well as persistence although no lesions are observed at these sites and the level of viral amplification are relatively modest.

Clearance of viral RNA from most of the tissues occurred relatively rapidly, but the rate of clearance was largely independent of the level of viral RNA. Cornified squamous epithelia of the skin and mouth contained the highest levels of viral RNA during the acute stage of infection, but the viral RNA in these tissues as well as other non-pharyngeal tissues was cleared faster than in pharyngeal tissues, in particular DSP and nasopharynx as well as MLN draining the region. Viral RNA persisted in pharyngeal tissues after 28 days p.i. in 10 of 14 animals examined, although the levels are low compared with those during acute infection. These data, together with the association of the presence of viral RNA in pharyngeal tissues, in particularly DSP, with the presence of infectious virus in OP-fluid samples as measured by infectivity in BTY cells, suggested that the target region involved in persistent infection in cattle is the pharyngeal tissue, in particular, DSP as well as nasopharynx (the dorsal part of the pharyngeal ceiling located above the soft palate). No clear explanations can be put forward for the difference in clearance of FMDV between pharyngeal tissues and non-pharyngeal tissues. The difference may be because of the cell type being infected in these tissues or by the presence of local factors such as cytokines, which may exert a profound effect on FMDV replication (Zhang et al., 2002). It is noteworthy that no significant histopathological changes have been reported to occur in this area even in acute infection (Alexandersen et al., 2001, 2002b; Salt, 1993, 1998). The epithelia at the DSP and the nasopharynx are highly specialized, non-cornified, stratified squamous epithelia, which are different from most of the surrounding epithelia. FMDV infection is usually highly cytolytic to target cells, but the presence of virus in epithelial cells of the soft palate not showing any significant lesions or cytopathic effect suggests that establishment of FMDV persistent infections in vivo may, in part, depend on the type of target cells infected (Alexandersen et al., 2002b, 2003b; Zhang & Kitching, 2000, 2001). This leads to the hypothesis that the epithelial cells in the pharyngeal region might be partially resistant to infection or the virus replication might be restricted in these cells by an, as yet, unknown mechanism, probably facilitated by the virus exploiting the host response to provide an intracellular milieu favourable for long term persistence of FMDV in contrast to the normally cytolytic, acute infection. Thus, it will be important to examine in more detail the potential role of epithelial cells in the pharyngeal region during the persistence of FMDV and to define the mechanisms by which the carrier status is established and maintained. Interestingly, there is no detectable virus or viral RNA (analysed in tissue samples) in pigs at 3 weeks after infection (Alexandersen et al., 2003b) although, FMDV was found in relatively high concentrations in soft palate, tonsil and pharynx in early infection (Alexandersen et al., 2001; Oleksiewicz et al., 2001). The mechanism for the reason why the pharyngeal region does not become persistently infected in pigs remains unknown.

In exploring possible non-pharyngeal sites of FMDV persistence, it was observed that tissues from non-pharyngeal regions and lymph nodes are not likely to be sites of persistence, although FMDV RNA can be found in MLN in a proportion of carrier cattle. This was probably because of drainage of the virus located in the pharynx. Based on dot blot analysis, it has been suggested that FMDV RNA may be associated with PBMCs even during persistent infection (Bergmann et al., 1996). However, by using quantitative RT-PCR, we have not been able to detect any detectable amounts of FMDV RNA in the PBMCs of carrier cattle and the levels during acute infection were very low and transient. Therefore, during the acute stage of FMDV infection virus circulates mainly/entirely as free virus in the bloodstream, but when antibodies are produced the virus is cleared within a few days and is no longer detectable in blood samples.

These quantitative studies of FMDV infection and replication in vivo provide a detailed description of the patterns of virus load and distribution associated with clinical and persistent infections. It appears that the extent of acute viral cytopathology can be most closely associated with the levels of virus replication in tissues having cornified squamous epithelia. The acutely infected animals had much greater levels of viral RNA compared with carrier animals, in which the levels of viral RNA were clearly much reduced and limited to particular tissues. The data suggest that during acute disease, the cornified squamous epithelia of the tongue and feet were the predominant cellular sites of active virus replication. During persistence, tight but not absolute control of virus replication/load was evident, indicating that a continuous low-level of replication persisted in non-cornified squamous epithelia of the dorsal soft palate and nasopharynx. It is clear that quantitative analysis of viral load in vivo is a valuable tool in order to fully understand the pathogenic steps of FMDV infection.


   ACKNOWLEDGEMENTS
 
We thank Vidhi Aggarwal for her excellent technical assistance; Colin Randall, Luke Fitzpatrick, Brian Taylor and Malcolm Turner for their assistance with the handling and management of experimental animals. This work was supported by the Department for Environment, Food and Rural Affairs (DEFRA), UK; Biotechnology and Biological Sciences Research Council (BBSRC), UK; and EU Contract QLK2-2002-01719.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 3 February 2004; accepted 17 May 2004.