Download Quantification and duration of Foot-and

Appendix 18
Quantification and duration of Foot-and-Mouth disease virus RNA in
bovine esopharyngeal fluid as determined by real-time RT-PCR
Zhidong Zhang , Ciara Murphy, Melvyn Quan, Jeanette Knight and Soren Alexandersen
Institute for Animal Health, Pirbright, Woking, Surrey, GU24 ONF, U.K.
Abstract: Foot-and-mouth disease virus (FMDV) load was quantitated in experimentally
infected cattle in order to improve knowledge about critical features of the initial virus-host
interactions which determine disease outcome. A quantitative real-time RT-PCR was used to
measure FMDV RNA in oesophageal-pharyngeal fluid (OP-fluid) samples from cattle
experimentally infected with type O FMDV by inoculation and contact. The dynamics of
FMDV load in OP-fluids exhibited remarkable similarities with viral replication patterns
observed in nasal and mouth swab samples for the first week of infection. Viral RNA was
recovered at 24 hours post infection (hpi), and rapid viral replication led to peak levels of
RNA viral load by 30 hpi to 53 hpi. The extent of virus declined after the peak. Complete
clearance of viral RNA occurred in some animals between 7 and 18 dpi. However, viral RNA
persisted in OP-fluids at detectable levels beyond 28 dpi in persistently infected cattle (socalled carriers) and was still detectable in some animals at 57 dpi when the experiments were
terminated. Viral RNA could not be detected in nasal and mouth swabs from FMDV carriers
and non-carriers at any time after 7-18 dpi. An association between the extent of virus
replication (growth rate) during acute infection and the outcome of infection (persistence or
non-persistence) could not be established. The most significant predictor of the outcome of
FMDV infection (persistence or non-persistence) was the extent of viral “clearance” (decay
rate) following peak levels. Animals with a slow decay rate became carriers while more rapid
clearance of viral RNA was characteristic of non-carriers.
Introduction
Foot-and-mouth disease virus (FMDV) causes a highly contagious viral disease of
domesticated and wild ruminants and pigs. Of considerable importance in the control of FMD
is the persistent infection that can occur following clinical or sub-clinical infection in both
vaccinated and non-vaccinated ruminants (so-called carriers). There is field evidence to
indicate that these carrier animals can precipitate new outbreaks of disease 2. A carrier is
defined as an animal from which live virus can be recovered for longer than 28 days after
exposure 9,11. Persistent FMDV is eventually eliminated from the carriers, but during the
period of persistence, there is considerable variation in the levels of virus recovery from
oesophago-pharyngeal fluid (OP-fluid) samples. On the other hand, persistent infection does
not occur in all infected ruminants, i.e. only a proportion of infected ruminants become
FMDV carriers 2. The mechanism for these phenomena remains to be fully understood
although the immune status of the animal prior to contact has been shown not to influence the
development of a carrier state.
A number of studies on experimentally infected cattle have unequivocally shown the
importance of pharyngeal area tissues in FMDV infection and replication during acute
disease or persistence 4,5,7,13. During persistence, infectious virus can be isolated from the OP-
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fluid samples2. However, the kinetics of viral RNA load in OP-fluids and its relationship with
the outcome of the infection (non-persistence and persistence) remains to be fully elucidated.
Intensive and quantitative analysis of FMDV RNA load is made possible by the recent
development of sensitive quantitative real-time RT-PCR 1,6,8. In this study the load of FMDV
RNA was quantified in OP-fluid samples from cattle experimentally infected with FMDV O
during the period of acute and persistent infection.
Methods
Animals - Twenty standard Compton steers (Holsteins) at 6-10 months of age were used and
divided into three groups in this study. Cattle in the first group (Group 1) and the second
group (Group 2) were infected with FMDV O UKG34/2001. Eight cattle in Group 1 were
infected by intradermolingual inoculation with 106.9 TCID50 and placed in pairs in 4 boxes.
The eight cattle in Group 2 were placed 4 and 4 in two boxes, with two cattle in each box
infected under the same conditions as the first group. Four cattle in the third group (Group 3)
were infected with FMDV O BFS1860 by intradermolingual inoculation of 107 TCID50. After
infection, inoculated and direct contact cattle were monitored for clinical signs of disease and
the rectal temperatures were recorded daily until 10 days after inoculation. Samples of OPfluid and nasal and mouth swabs were collected from before the start of the experiment
(negative controls) and after infection. OP-fluid samples were collected using a probang cup 11
and diluted in an equal volume of Eagle’s-HEPES medium (pH7.2) containing 5% FCS and
stored at -80°C until required. The infectivity of OP-fluid samples was determined by
inoculation of monolayers of primary bovine thyroid (BTY) cells, essentially as described by
Snowdon (1966)10. Nasal and mouth swabs were placed in 1 ml of Trizol (Life Technologies,
UK) and stored at -80°C.
Nucleic acid purification - 200 µl of samples were mixed with 300 µl of MagNa Pure LC
total nucleic acid lysis buffer (Roche, UK). Total RNA was extracted using MagNa Pure LC
total nucleic acid isolation kit (Roche, UK) with an automated nucleic acid robotic
workstation according to the manufacturer's instruction and stored at -80°C until used. The
concentration of the RNA was determined by spectrophotometry.
Quantification of viral RNA by real-time quantitative RT-PCR analyses - The level of
viral RNA in samples was quantified by a real-time RT-PCR as described previously 3,8. 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, UK) according to the manufacturer's directions. RNA was resuspended in DEPCtreated water and quantified by spectrophotometry.
Calculation of the rates of virus growth and decay during infection - Initial viral
replication rates were calculated by using the exponential growth rate equation: γ = (lnY1lnY2)/(t1- t 2), where Y1 and Y2 are the virus load values at times t1 and t2, respectively. The
virus load doubling time, T2, in hours was calculated by using the equation T2 = (ln2)/γ. Rates
of viral decay were calculated by using the decay rate equation (α) = (lnY1-lnY2)/(t1-t2). The
viral decay half-life, T1/2, in hours was calculated by using the equation T1/2 = (ln2)/α. Viral
decay rates and T1/2 were calculated with the steepest interval of the decay curve following
the peak data point.
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Statistical analyses - Statistical analyses were performed on log10-transformed FMDV RNA
values, or ln values in the case of viral growth and decay rates.
Results
Dynamics of viral RNA levels in oesophageal-pharyngeal fluid samples during the acute
stage - In order to characterise the early course of viral RNA kinetics in OP-fluids following
infection with FMDV, we measured the viral RNA level in OP-fluid samples collected twice
a day during the first 2-3 days following inoculation, followed by daily measurements until 7
dpi. At 6 hpi, three of 12 inoculated cattle with FMDV O UKG (Group 1 and Group 2)
manifested FMDV RNA, by 24 hpi all inoculated cattle had a quantified level of FMDV
RNA ranging from 6.89 to 9.54 log10 copies/ml (average 7.97 ± 0.99; median 7.79) (Fig. 1A
and 1B). Viral RNA levels then peaked at 30 or 53 hpi. The maximum level of FMDV RNA
varied from 7.52 to 9.54 log10 copies/ml (average 8.55 ± 0.66; median 8.14). Thereafter,
FMDV RNA levels decreased by various extents. The contact cattle in Group 2 had a similar
kinetic pattern of viral RNA in OP-fluid samples (Fig. 1C). However, Viral RNA levels did
not peak (average 8.21 ± 0.74 log10 copies/ml; median 8.06) until 5 days after contact. A
similar pattern of FMDV RNA level was also seen in OP-fluids of Group 3 cattle infected
with FMDV O BFS (Fig. 1D). The peak levels of FMDV RNA (average 7.95 ± 0.92 log10
copies/ml; median 7.28) was lower than those in cattle infected with FMDV O UKG, but no
statistically significant differences were identified (non-parametric test, p > 0.05).
The kinetic patterns of viral RNA in the OP-fluid samples exhibited similarities with the
dynamics of FMDV RNA observed in nasal and mouth swabs during the first weeks of
infection (more detailed results with regard to these will be given in the presentation by Soren
Alexandersen at this meeting). The peak levels of viral RNA in OP-fluid samples were not
statistically significantly different from those in nasal and mouth swabs (Fig. 2, nonparametric test, p > 0.05).
Duration of viral RNA in oesophageal-pharyngeal fluids - Although there is no significant
difference in the peak level of viral RNA amongst nasal, mouth swabs and OP-fluid samples,
duration of viral RNA in these samples is significantly different. Representative results from
Group 1 cattle are shown in Fig. 2. It has been demonstrated that RNA levels in serum
declined much faster and become undetectable after 4 dpi when antibodies became
detectable3 (more detailed results with regard to these will be given in the presentation by
Soren Alexandersen at this meeting). In general, virus RNA in nasal and mouth swabs were
detected for a longer period than virus RNA in serum but shorter than that in OP-fluid
samples. Virus RNA was consistently detectable for up to 7 days in nasal swabs and
consistently for about 5 days in mouth swabs, but 6 out of 12 mouth swabs from inoculated
cattle with FMDV O UKG continued to have detectable levels of virus RNA for some days
longer and a single animal (a carrier) had virus RNA in the mouth swabs up to 18 days after
inoculation. OP-fluid samples were consistently positive up to around 7-10 days after
inoculation, after that time 13 out of 20 cattle were still positive and 8 cattle was positive
beyond 28 dpi and up to 57 dpi. In Group 1 carrier animals, levels of viral RNA in OP-fluid
samples varied from 4.72 log10 copies/ml at 28 dpi to 5.15 log10 copies/ml at 41 dpi. In Group
2 FMDV RNA load varied from 5.54 ± 1.12 log10 copies/ml at 30 dpi to 3.29 ± 0.84 log10 at
44 dpi. Of Group 3 animals, the viral RNA load varied from 5.13 ± 1.65 log10 copies/ml at 30
dpi to 5.15 log10 copies/ml at 39 dpi. All these OP-fluid samples from carriers contained the
infectious virus as determined by inoculation onto monolayers of BTY cells. At this time, all
other samples were consistently negative.
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The extent of reduction of viral load after peak level in oesophageal-pharyngeal fluids is
a critical determinant of the outcome of FMDV infection - To evaluate the possibility that
the temporal pattern of OP-fluid viral RNA in the early course of infection is related to the
outcome of the infection, cattle were divided into carriers and non-carriers according to the
persistent status (persistent and non-persistent) 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 (γ)
and standard deviation (SD) of virus growth and the corresponding doubling time, T2 in hours
(described in Methods). Growth slopes, in ln units, were calculated by using the initial two
quantifiable data points for each animal. As shown in Fig. 3, the mean viral growth rate in
carriers (γ = 0.24 ± 0.19; T2 = 2.9 hours) was similar to those in non-carriers (γ = 0.20 ± 0.14;
T2 = 3.45 hours). No statistically significant differences were identified (non-parametric test,
p > 0.05).
To determine whether the extent of reduction of viral load after peak levels in OP-fluid
samples is correlated with the outcome of the infection, this phase of the FMDV infection
was analysed by calculation of the slopes of early viral decay and virus load half-life in OPfluids, T1/2 in hours (as described in Methods). Decay slopes, in ln units, were calculated from
the steepest interval of the decay curve following the peak for each animal. Carriers in Group
1 cattle had a lower decay rate (α = 0.03; T1/2 = 23.1 hours) than that in non-carriers (α =
0.11 ± 0.01; T1/2 = 6.3 hours). A similar phenomenon was also seen in OP-fluid samples in
contact cattle of Group 2, in which the carriers had a lower decay rate (α = 0.02 ± 0.01; T1/2 =
34.7 hours) than that in non-carriers (α = 0.08 ± 0.05; T1/2 = 8.7 hours). Moreover, carriers in
Group 3 also had a mean decay rate (α = 0.04 ± 0.01; T1/2 = 17.3 hours) which was lower
than that in the non-carriers (α = 0.06 ± 0.01; T1/2 = 11.6 hours). Given these data as a whole,
the mean decay rate (α = 0.03 ± 0.02; T1/2 = 23.1 hours) in carriers is significantly different
from that in the non-carriers (α = 0.09 ± 0.06; T1/2 = 7.7 hours; non-parametric test, p < 0.05)
(Fig. 3). These data suggest that the early decay rate of FMDV RNA in OP-fluid samples is a
critical determinant of the duration of FMDV persistence. Viral decay rate in nasal and mouth
swabs (based on Group 1) was higher than that in OP-fluid samples and no significant
difference was identified between carriers and non-carriers (data not shown).
Relationship between virus replication during the acute phase and the duration of virus
persistence - The animals in this study cleared virus at widely varying times. In order to
ascertain whether the characteristics of early viral replication influence the outcome of
disease course, the course of acute FMDV infection was divided into three phases: pre-peak,
peak and post-peak. The extent of early virus growth might influence the resulting host-virus
equilibrium. An association was explored between early viral levels (defined as levels at 24
hours) and peak levels in OP-fluid samples collected from Group 1 animals. There was no
correlation among these parameters (r = 0.28, p > 0.05). There also did not appear to be any
correlation between early virus growth rates and subsequent events (persistent and nonpersistent). Similar results are also seen in Group 2 and 3 animals. Peak viral load in OP-fluid
samples might reflect the extent of cell targets available for FMDV replication. Interestingly,
the highest peak viral load was observed in animal UO32, which thereafter exhibited control
of virus replication and clearance of the virus after 15 days. No correlation was found
between viral peak and subsequent events (persistent and non-persistent) because there is no
significant difference in the viral peak levels between carriers (8.38 ± 0.38) and non carriers
(8.30 ± 0.33) (non-parametric test, p > 0.05). The post-peak level of viral RNA decline in
OP-fluid samples might represent the best measure of host ability to clear virus, as early viral
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RNA decline in OP-fluid samples was correlated with the duration of virus persistence. The
slower the viral RNA declined early after the peak, the longer the virus persisted in OP-fluid
samples.
Discussion
This study has quantitatively analysed the dynamics of FMDV RNA in bovine OP-fluid
samples. In order to test the hypothesis that the events occurring shortly after infection
dictated subsequent infection outcome (persistent and non-persistent), we performed frequent
monitoring of the levels of viral RNA in bovine OP-fluid samples following FMDV O
UKG2001 infection. We demonstrated that all OP-fluid samples from carriers which had a
quantifiable level of viral RNA as measured by the real-time RT-PCR, were positive in the
virus isolation test. Previous studies has also shown that viral RNA copy numbers measured
by the real-time RT-CPR correlated well with virus infectivity 1,3. Therefore, the level of viral
RNA could be a very useful indicator of the profiles of viral load.
Following the peak viral load in OP-fluid samples, the 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. Viral variation seems unlikely to be the cause of these
differences. The differential emergence of viral variants with different replicative capacity
within the first week of infection seems unlikely among animals that received identical
inocula of FMDV O UKG2001. 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 is caused by a difference in ability to clear virus or caused by a
continuing viral 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 4,5. During persistence,
viral RNA was detected in bovine pharyngeal epithelia samples7 and recently localised in
epithelial cells of the bovine pharyngeal area 13. 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 shown to persistently harbour FMDV did not show any cytopathic
changes for many weeks which further support this theory 12. 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 specialised cellular site. Therefore, it
may be considered especially important to define those cellular factors and mechanism
involved in the regulation of virus expression and clearance in these cells. In fact such
knowledge would also be extremely helpful in defining selective approaches to control
FMDV in persistently infected animals.
This study highlights the role of early events in the establishment of the 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
We thank 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.
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Carriers
A: Group 1 animals (n=8)
Carriers vs non-carriers
Non-carriers
B: Group 2 contact animals (n=4)
Carriers vs non-carriers
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Fig.1 Kinetics of FMDV RNA in oesophageal-pharyngeal fluids. FMDV RNA loads are
expressed as log10 copy number per ml. Animals are divided into carriers and non-carriers
according to persistent status.
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Carriers
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Fig.2. Duration of FMDV RNA in oesophageal-pharyngeal fluid samples, nasal and mouth
swabs collected from Group 1 cattle infected with FMDV O UKG2001. Animals were
divided into carrier and non-carriers as described in Methods. The viral RNA levels were
quantified by real-time RT–PCR and expressed as log10 copy number per ml.
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Doubling time
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Fig 3 Virus growth rate and decay slopes in oesophageal-pharyngeal samples. There are
statistically significant differences in the decay rate value (α) and half-life (T1/2) between
carriers (n=8) and non-carriers (n=9) (non-parametric statistics, p <0.05).
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