Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
Soren Alexandersen
soren.alexandersen{at}bbsrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Bovine pharyngeal tissue has been identified as a primary site for FMDV infection in vivo (Burrows et al., 1971; McVicar & Sutmoller, 1969
). Viral RNA has been detected in pharyngeal tissues (Prato Murphy et al., 1994
) and has been localized in epithelial cells of the pharyngeal tissues of carrier cattle (Zhang & Kitching, 2001
). These studies indicate that FMDV in the pharyngeal area tissues could be a source for the virus present in oesophagealpharyngeal fluids (OP-fluids). During acute disease and persistence, infectious FMDV can be isolated from OP-fluid samples, but the amounts of virus in OP-fluid samples are generally low during persistence. During the period of persistence, there is considerable variation in the levels of virus recovery from OP-fluid samples (reviewed by Alexandersen et al., 2002a
). However, a great deal remains to be learned about quantitative aspects of viral RNA load in OP-fluids and its relationship with the outcome of the infection (persistence or non-persistence). Quantitative analysis of FMDV RNA load is made possible by the recent development of sensitive, quantitative real-time RT-PCR (Alexandersen et al., 2002b
; Oleksiewicz et al., 2001
; Reid et al., 2002
; Zhang & Alexandersen, 2003
). Studies have shown that viral RNA copy numbers measured by real-time RT-PCR correlate well with virus infectivity (Alexandersen et al., 2001
, 2002b
, 2003a
, b
; Zhang & Alexandersen, 2003
). Therefore, the level of viral RNA could be a very useful indicator of the profiles of viral load.
In this study the load of FMDV RNA was quantified in OP-fluid samples from cattle experimentally infected with FMDV type O during the period of acute and persistent infection. The results suggest that the extent of reduction of viral RNA in OP-fluid samples immediately following peak levels is a critical determinant of the outcome of FMDV persistence.
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METHODS |
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Assay for virus.
The infectivity of OP-fluid samples was determined by inoculation of monolayers of primary bovine thyroid (BTY) cells, as described by Snowdon (1966).
RNA extraction.
For automated RNA extraction, 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 the MagNa Pure LC total nucleic acid isolation kit (Roche) with an automated nucleic acid robotic workstation (Roche), according to the manufacturer's instructions, and stored at -80 °C until used as described previously (Alexandersen et al., 2003a).
Real-time quantitative RT-PCR assay for detection of viral RNA.
The level of viral RNA in samples was quantified by real-time RT-PCR as described previously (Alexandersen et al., 2002b, 2003a
; Reid et al., 2002
; Zhang & Alexandersen, 2003
). For the generation of standard curves, standard viral RNA was generated from plasmid pT73S containing full-length FMDV (kindly provided by Dr Andrew King, Institute for Animal Health, UK) by in vitro transcription using a commercially available T7 RNA polymerase kit (Ambion), according to the manufacturer's instructions, as described previously (Zhang et al., 2002
). RNA was resuspended in RNase-free water and quantified by spectrophotometry.
Calculation of the rates of virus growth and decay.
Initial virus replication rates were calculated by using the exponential growth rate equation =(lnY1-lnY2)/(t1-t2), where Y1 and Y2 are the virus load values at times t1 and t2 (in hours), respectively. The virus load doubling time, T2 (in hours), was calculated by using the equation T2=(ln2)/
. Rates of viral decay or clearance were calculated by using the decay rate equation
=(lnY1-lnY2)/(t1-t2), where Y1 and Y2 are the virus load values at times t1 and t2 (in hours), respectively. The viral decay/clearance half-life, T1/2 (in hours), was calculated by using the equation T1/2=(ln2)/
. Viral decay rates and T1/2 were calculated with the steepest interval of the decay curve within 2 weeks p.i. following the peak.
Statistical analyses.
Statistical analyses were performed by using a non-parametric test (MannWhitney test using MINITAB release 12.21 software) on log10-transformed FMDV RNA values, or ln values in the case of viral growth and decay rates. P <0·05 was considered statistically significant.
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RESULTS |
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The extent of viral RNA decay after peak levels is associated with the outcome of virus persistence
To evaluate the possibility that the temporal pattern of viral RNA in OP-fluid samples in the early course of infection is related to the outcome of the infection (persistent or non-persistent), the cattle were divided into carriers and non-carriers, according to persistence status, and then the initial virus growth rate and subsequent decay slopes were calculated. Early virus replication in individual animals was assessed by estimation of the mean rate ()±SD of virus growth and the corresponding doubling time, T2, in hours (described in Methods). Growth slopes were calculated by using the two initial quantifiable data points for each animal. As shown in Fig. 3
, the mean viral growth rate in carriers [
=0·30±0·28, T2=3·84 h, n=11 (no suitable samples were available from one animal to do the calculations and this animal was therefore excluded)] was slightly higher than those in non-carriers (
=0·26±0·21, T2=4·11 h, n=12), but no statistically significant differences were identified (non-parametric test, P=0·93). Therefore, the outcome of the infection (persistent or non-persistent) appears to be independent of early virus growth rates.
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DISCUSSION |
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To test the hypothesis that the events occurring shortly after infection dictated subsequent infection outcome, frequent monitoring of the levels of viral RNA in bovine OP-fluid samples following FMDV type O infection was performed. We demonstrated that following the peak viral load in OP-fluid samples, the early rate and extent of viral decay was significantly different between carrier and non-carrier animals receiving identical inocula. What determines the magnitude of the reduction of RNA load in OP-fluid samples following the peak load is not fully understood. The differential emergence of viral variants with different replicative capacity within the first week of infection seems unlikely among animals that received identical inocula. Thus, this observation may simply reflect variations in the kinetics of the host response to infection, i.e. clearance of virus or continuing ability to support replication. We are now investigating the quantitative aspects of early events in the host response to infection in cattle, sheep and pigs to determine whether the observed differences in decay rate are caused by differences in the ability to clear virus or by differences in the occurrence or level of continuing virus replication.
Virus RNA persisted in OP-fluids for a longer period than in other sample types. The reason for this phenomenon remains to be fully understood. Bovine pharyngeal tissue has been identified as a primary site for FMDV infection and replication in vivo (Burrows et al., 1971; McVicar & Sutmoller, 1969
). During persistence, viral RNA has been detected in bovine pharyngeal tissues (Prato Murphy et al., 1994
) and has been recently localized in epithelial cells of the bovine pharyngeal area (Zhang & Kitching, 2001
). These studies of FMDV infection in experimentally infected cattle have unequivocally shown the importance of pharyngeal area tissues in virus infection and replication. The epithelial cells isolated from pharyngeal tissues of FMDV infected cattle and cultured in vitro have been shown to persistently harbour FMDV and they did not show any cytopathic changes for many weeks; both observations further emphasize the importance of these cells (Zhang et al., 2002
). This leads to the hypothesis that the presence of viral RNA and infectious virus in OP-fluids during persistence is because FMDV may be able to continuously replicate in such a specialized cellular site without being efficiently cleared. Therefore, it may be especially important to define those cellular factors and mechanisms involved in the regulation of virus replication and clearance in these cells. In fact, such knowledge would also be extremely helpful in defining selective approaches to the control of FMDV in persistently infected animals.
The experiments described here included experimental infection with FMDV O UK 34/2001 and O BFS1860, and resulted in the development of the carrier state in 50 % of the 24 cattle studied for more than 28 days p.i. Other studies using similar conditions (Z. Zhang & S. Alexandersen, unpublished) showed that 0/7 cattle infected with FMDV O SKR 1/2000 in two separate experiments became carriers, while all nine cattle infected with C Oberbayren in two separate experiments became carriers. The O SKR 1/2000 virus only caused mild clinical disease in cattle and the virus was rapidly cleared from all sites, including the pharynx, while C Oberbayren caused severe clinical disease and was cleared slowly from the pharyngeal region with a pattern consistent with the carrier animals described in this study (Z. Zhang & S. Alexandersen, unpublished). Taken together, these findings suggest that the establishment of the carrier state is an early event and that the efficiency or frequency may be dependent on both the virus strain and the host. Furthermore, as the development of the carrier state is associated with a slower clearance rate immediately after the peak viral load, it may be possible to use these findings for practical disease control, i.e. to predict the risk of carriers occurring under certain epidemiological conditions. As discussed above, the exact mechanisms involved in FMDV persistence or clearance in vivo are not clear, but are likely to include both viral and host factors. Levels of circulating antibodies failed to correlate with the development of carriers. As the kinetics of viral load and clearance differed between subsequent carriers and non-carriers very early following infection, the fine details of the innate immune response may prove to be an important host factor.
This study highlights the role of early events in the establishment of persistent FMDV infection. The extent of reduction of viral load after the peak is a critical determinant of the outcome of FMDV infection. Elucidation of the mechanisms that account for these observations will provide insight into the pathogenesis of FMD and may have important practical consequences for the development of an effective vaccine for prevention of carriers.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 July 2003;
accepted 15 October 2003.