Content uploaded by Andrzej Fitzner
Author content
All content in this area was uploaded by Andrzej Fitzner on Jan 20, 2015
Content may be subject to copyright.
Bull Vet Inst Pulawy 55, 581-586, 2011
DETECTION OF RHD VIRUS
BY A REAL-TIME REVERSE TRANSCRIPTION PCR
ANDRZEJ FITZNER, WIESŁAW NIEDBALSKI, ANDRZEJ KĘSY,
AND
GRAŻYNA PAPROCKA
Department of Foot-and-Mouth Disease, National Veterinary Research Institute,
98-220 Zduńska Wola, Poland
andrzej.fitzner@piwzp.pl
Received: October 4, 2011 Accepted: November 22, 2011
Abstract
A real-time RT–PCR method for the rapid detection of the rabbit haemorrhagic disease virus (RHDV) in the liver and
serum samples of rabbits was described. A primer set that targets 3’ part of VP60 gene and TaqMan probe specific for the conserved
region in RHDV genome was used in the method. The assay was able to detect genetic material in rabbits infected with classic
RHDV as well as RHDVa variant. RNA of both haemagglutinating and non-heamagglutinating strains were also detected in samples
with different virus strains. The detection limit of RHDV RNA by rRT-PCR was 10
-7
. The method can provide quantitative and
qualitative information and is more sensitive and faster than the conventional RT-PCR. Therefore, it seems to be a valuable tool to
complete the routine diagnostic procedure in RHD diagnosis.
Key words:
rabbit haemorrhagic disease virus, detection, real-time PCR.
For nearly thirty years rabbit haemorrhagic
disease (RHD) has threatened the rabbit breeding and
meat production in the world. The fatal disease with
extremely high levels of morbidity and mortality of
susceptible wild and domestic rabbits of the species
Oryctolagus Cuniculus was recognised in 1984 in China
(16). In Europe, highly infectious plaque, of unusual
epidemiological, clinical, and pathological findings
(named “Mallatia X”) occurred in 1986 in Italy. At the
same time, apparently similar haemorrhagic syndrome
(EBHS) was recognised in hares (2, 18). In Poland, the
first outbreaks of RHD were officially reported in 1988
(9). A common feature found during RHD epidemics,
regardless of geographical location, is that young
animals, usually less than two months, were immune (11,
12).
The causative agent, rabbit haemorrhagic
disease virus (RHDV), family Caliciviridae, has a
single-stranded positive sense genome of 7.5 Kb (17).
RHDV strains are characterised by a high ability to
haemagglutinate human red blood cells, although non-
heamagglutinating phenotypic variants were also
described (5, 10). All known strains of RHDV belong to
one serotype. RHDV does not reproduce in cell culture
and the main method for its multiplication is infection of
sensitive rabbits (12). RHDV is very stable under
different environmental conditions. Small, non-
enveloped viral particles (about 35 nm) are resistant to
ether and chloroform. Under experimental conditions,
virus stored in frozen or lyophilised homogenates can
survive for many years without reducing its infectivity,
antigenicity, and haemagglutination activity (6, 19, 20).
Initially, genetic characterisation of the RHDV
strains demonstrated a considerable stability of virus
genome with low percentage of mutation. Based on
antigenic and genetic characteristics, newly RHDV
subtype named RHDVa was detected in 1998 (4, 21). In
addition, non-pathogenic calicivirus (RCV), closely
related to RHDV, was isolated in the gastrointestinal
tract of rabbits (3). In most cases, conventional
virological and serological tests (HA, HI, different types
of ELISA) allow the confirmation of clinical diagnosis
of RHD. For detection of RHDV genetic material,
conventional PCR methods were evaluated using
numerous sets of nucleotide primers, that amplified the
vp60 gene (4, 11, 13).
The aim of this study was to determine the
diagnostic value of a real-time reverse transcription
assay for the detection of RHDV RNA in rabbits from
RHDV infected animals.
Material and Methods
Sample origin. The 23 archival Polish RHDV
isolates, including non-haemagglutinating phenotypic
variants and RHDVa subtype strains were collected in
our laboratory from 1988 to 2006. Among them, there
were KGM and SGM strains isolated in 1988 from the
first officially confirmed RHD outbreak in Poland (9),
and KGM HA positive isolate, which has been used as a
vaccine strain until 2008. Additionally, French strain
582
(PLF 83/92-352), passage of German HA-negative strain
Frankfurt, and passage of Czech strain V-351 (kindly
provided from the Centre National d’Etudes Vétérinaires
et Alimentaires (CNEVA) in Ploufragan (France) and
the University of Szczecin, Poland) were used (Table 1).
RHD virus was extracted from the liver taken from
infected rabbits and stored frozen at -18 - -26°C. A total
of 2 g of liver tissue was homogenised in 8 ml of PBS,
purified with chloroform (10%), and centrifuged at
7,000 g for 10 min. A part of the homogenates was
stored frozen after mixing with glycerol (1:1) or in a
lyophilised form at final concentrations 10% w/v. All
samples were confirmed positive for the presence of
RHD virus by ELISA, heamagglutination test, and
conventional reverse transcriptase polymerase chain
reaction. Additionally, the following material was tested:
seven liver specimens of healthy rabbits taken from the
slaughterhouse between 2008 and 2011 (two non
vaccinated, 3-month-old New Zealand white rabbits, five
mixed-race rabbits, one mixed-race rabbit f with hepatic
coccidiosis) (Table 1), seven serum samples of
seropositive convalescent rabbits taken after 7 d, 6
weeks, and 7 months after beginning of two RHD
outbreaks (in 1994 and 2004), and serum samples of
vaccinated and control (non vaccinated) rabbits taken
prior and after experimental infection (two control
rabbits survived the challenge and exhibited a maximum
body temperature rise to 41.7-41.3°C at 3-5d p.i. (Table
3). Moreover, bluetongue virus (BTV) RNA extracted
from EDTA blood sample (serotype 8) was used.
Nucleid acid extraction. A hundred microlitres
of the liver tissue homogenate or serum samples was
used. The total RNA was extracted in a volume of 35 µl
using the RNeasy Mini Kit (Qiagen) according to
manufacturer’s instruction. Extracted undiluted RNA
and ten-fold serial dilutions were used for the real time
PCR, or in reverse transcription for conventional RT-
PCR.
Real-time reverse transcription (rRT-PCR).
The probe (6986-7010): FAM-CCA
ARAGCACRCTCGTGTTCA ACCT-TAMRA, and a
pair of oligonucleotide primers P7vp60 (6941-6961)
ACYTGACTGAACTYATTGACG, P8 vp60 (7044-
7022) TCAGACATAAGAAAAGCCATTGG were used
according to Gall et al. (8). The assay was performed in
Real Time 7300 thermal cycler (Applied Biosystems) as
a one-step reaction using the Quanti Tect Probe PCR Kit
(Qiagen). The reaction mixture at the volume of 25 µl
contained 5 µl of extracted RNA, 12.5 µl of 2x Quanti
tect Probe RT-PCR Master Mix, 0.25 µl of Quanti Tect
RT Mix, 0.5 µl (2.5 pmol) of Taqman probe, 1.0 µl of
forward primer and 1.0 µl of reverse primer, both at
concentration of 10 pmol, 1.25 µl of MgSO
4
, and 3.5 µl
of H
2
O. The cycling conditions were as follows: I -
reverse transcription - 50°C/30 min; II - reverse
transcription inactivation and Taq DNA polymerase
activation - 94°C/2 min; III – 45 cycles of PCR -
94°C/30 s, 55°C/45 s, 68°C/45 s (8). The reporter dye
(FAM) was measured at the stage of annealing in the
exponential phase of the amplification plot of each cycle.
After completion of the PCR run, threshold – crossing
values (C
T
) were established automatically at 0.2 level.
Reverse transcription (RT) and conventional
PCR. The total RNA was incubated for 15 min in 18-
26°C with the reaction mixture. The synthesis of cDNA
was performed at 42°C for 60 min in a 60 µl reaction
using 1 µl of oligo dt15 primer (Promega), PCR
nucleotide mix, and AMV reverse transcription enzyme
(Promega). The PCR conditions were as follows:
denaturation for 3 min at 94°C, 35 cycles of
amplification (1 min at 94°C, 1 min at 55°C, 1 min at
72°C), and final elongation 10 min at 72°C. The 510 bp
amplicon of VP60 gene was obtained with the use of
oligonucleotide primers: P1(5182)
5’GAGCTCGAGCGACAACAGGC, P2 (5692)
5’CAAACACCTGACCCGGCAAC according to Guittré
(13). The primers were synthesised in the Institute of
Biochemistry and Biophysics (Warsaw). RNAase free
water was used as a negative cDNA template.
Results
The mean C
T
value for undiluted RNA of 26
samples extracted from liver homogenates of RHDV
infected rabbits was established at the 21
st
cycle
(threshold line 0.2). In 22 samples, C
T
values ranged
from 16.86 to 22.95. In this group, four of 26 samples
revealed C
T
values significantly higher; they ranged from
27.4 to 31.2. All analysed liver samples of infected
rabbits diagnosed positive in HA or ELISA were
recognised as positive by rRT-PCR, independently from
the genetic subtype or haemagglutination characterstics
(Table 1). For series of RNA dilution of the seven
RHDV strains, a mean C
T
values from 17.62 at undiluted
samples to Ct 40.01 at 10
-7
dilution were obtained. At the
highest dilution (10
-7
), Ct values fluctuated around 40,
ranging from 36.5 in one case to over 45 in the others
(Table 2). An example of rRT-PCR results of KGM
RHDV vaccine strain isolated from the liver of
experimentally inoculated rabbit (passage 5 from 2007)
is presented in Fig.1. The C
T
values of RNA extracted
from the liver of two healthy rabbits (L15/IV/2008 and
L8/RI/2008) and rabbit with hepatic coccidiosis (B
2011) were outside the 45 cycle. In the liver of four
rabbits rRT-PCR test demonstrated C
T
values near 40,
and in one case (L3/RI/2008) C
T
value was 32 (Table
1).
The mean C
T
value for RNA of serum samples
taken from infected rabbits, or animals suspected of
infection corresponded to 41 (Table 3).
In sample 6/8 taken from two rabbits, about a
week after disease started, C
T
of 35 was demonstrated. A
similar result was obtained for serum of control rabbit,
which survived 10 d after the experimental infection and
its maximum body temperature was 41.7°C on day 3
post challenge. No amplification RNA RHDV was
detected with BT virus sample.
583
Table 1
C
T
values of RHDV strains obtained by rRT-PCR assay after RNA isolation from liver samples
RHDVstrain
Antigenic type HA titer C
T
(The mean of four
repetitions)
KGM 1988 (passage 5/2007) RHDV 10,240 17.67
17.99
SGM 1988 (passage 2/1991) RHDV 2,560 27.38
PD 1989 RHDV 5,120 18.17
BLA 1994 (lyoph). RHDV Negative 21.83
MAL 1994 (lyoph). RHDV 2,560 22.58
PRB 1995 RHDV 10,240 16.68
17.21
BDG 1/1996 RHDV 2,560 18.08
BDG 2/ 1997 RHDV 160 19.97
BDG 3/1997 RHDV 2,560 19.20
BDG 4/1998 RHDV 20,480 21.14
GSK 1998 (lyoph.) RHDV 5,120 19.60
PIA 1999 RHDV 10,240 22.20
POZ 1999 RHDV 5,120 21,19
ZD0 2000 RHDV 2,560 27.61
SIZ 2002 RHDV 2,560 22.95
ZDU 2003 RHDVa 10,240 16.86
OPO 2004 HA neg. negative 17.86
GRZ 2004 RHDVa 20,480 17.71
ROK 2004 RHDVa 20,480 19.64
CB 2005 RHDVa 10,240 18.60
KRY 2005 RHDVa 2,560 31.19
ZKA 2005 RHDVa 5,120 19.78
DCE 2006 RHDVa 160 18.20
V-351 (lyoph.) RHDV 1,280 28.03
Frankfurt HA neg. negative 18.75
PLF 83/92-353 (lyoph.) RHDV 10,240 22.48
B 2011 (coccidiosis) negative negative undetected (>45)
Liver of healthy rabbits
L3/RI/2008
L13/RII/2008
L5/RIV/2008
L15/RIV/2008
L7/RI/2011
L8/RI/2011
L6/RII/2011
negative
negative
negative
negative
negative
negative
negative
32.35
40.79
40.04
undetected (>45)
40.99
undetected (>45)
40.75
Table 2
C
T
values of diluted RHDV RNA obtained by rRT-PCR
RHDV strain
(RNA isolation
number)
RNA dilution
10
-
1
10
-
2
10
-
3
10
-
4
10
-
5
10
-
6
10
-
7
KGM (1)
(2)
20.04 23.67
22.00
28,2
30,77
27.64
34,02
31.37
37,42
34.76
37.92
GRZ (RHDVa) 19.78 24.69 29.59 33.18 37.03 40.33
ROK (RHDVa) 19.33 21.85 24.44 25.39 29.17 31.78 36.47
DCE (RHDVa) 19.59 23.28 28.89 33.47 34.03 37.8 40.96
Frankfurt 18.4 21.56 25.38 28.62 32.63 35.43 38.98
ZDU (RHDVa) 18.25 21.77 24.69 27.60 31.70 35.00 37.54
PRB (1)
(2)
19.04
18.44
23.09
22.71
27.84
26.28
32.74
30.10
35.60
33.07
40.05
36.17
undet. (>45)
39.74
584
Table 3
C
T
values of RNA isolated from rabbit serum obtained by rRT-PCR assay.
Serum samples collected from rabbits at various time points since the disease outbreak, or after experimental
vaccination and inoculation test
Sample origin Serum number HI antibody titer C
T
Sera 2, 3, 4, and 8 - 6 weeks
after RHD outbreak in
Małogoszcz (MAL), 1994.
Serum 10 - 7 months after
RHD outbreak in Małogoszcz
1994.
S2
S3
S4
S8
S10
1,280
1,280
2,560
640
640
42.09
41.80
42.99
43.00
41.58
Serum samples – 7 d after
RHD outbreak in Opole
(OPO), 2004.
S6/8
S10
5,120
160
35.71
41.31
Serum of control rabbits (non-
vaccinated) - survived 10 d p.i
(vaccine control test)
S1 – 0 d
S1a – 10 d p.i
S2 - 0 d
S2a – 10 d p.i
<10
2,560
<10
1,280
40.2
35.33
41.54
39.47
Serum of vaccinated rabbit
(vaccine control test)
S1 – 14 d p.v
S1a – 10 d p.i
1,280
1,0240
40.69
41.50
Using conventional RT-PCR and the set of
specific primers flanking 5’ portion of RHDV capsid
structural protein, the viral RNA was detected in 26 liver
homogenates of rabbits infected with the analysed
RHDV strains. The 510 bp fragments of KGM isolate
were detected at RNA dilution from 10
-1
to 10
-5
(Fig. 2).
The agarose gel analysis of PCR products demonstrated
the presence of 510 bp fragments in two liver
homogenates (L3/RI/2008 and L5/IV/2008) of healthy
rabbits with corresponding mean C
T
values of 32 and 40,
respectively. No 510 bp fragment was detected in S1
serum sample taken 10 d post challenge (data not shown)
from control rabbits, which survived the experimental
infection
Discussion
The aim of the presented study was to assess the
potential use of the single one-step TaqMan rRT-PCR
method for virological diagnosis of RHD. Although
international diagnostic methods have not been validated
and there is no uniform pattern of RHD virus, many
serological and virological tests are currently used in
routine diagnosis. For virological purpose, different
ELISAs with specific monoclonal or polyclonal
antibodies, haemagglutination assay (HA), direct
immunoflurescence, and immune electron microscopy
are used (1). In addition, several RT-PCR assays were
developed and evaluated for the detection of RHDV
based on nucleotide sequences of different genome
fragments (3, 7, 11, 13, 14).
In recent years, various real-time PCR methods
have been implemented and applied to the diagnosis of
human and animal diseases (15). Real-time RT-PCR
(rRT-PCR) offers certain advantages over conventional
RT-PCR. It avoids the use of agarose gel
electrophoresis, therefore decreasing the risk of
contamination, and is suitable for large scale testing and
automation.
Fig. 1. Amplification plots of undiluted and diluted RNA
samples (log
10
dilution ) of KGM RHDV strain by rRT-PCR.
From left to right: RNA undiluted, 10
-2
, 10
-4
, 10
-5
, 10
-6
, 10
-7
,
negative control (H2O).
Fig. 2. Detection of amplicons 510 bp by RT-PCR method
using undiluted and diluted RNA samples extracted from the
liver of experimentally infected rabbit (strain KGM RHDV).
From left to right: DNA molecular weight marker Promega) –
1,000, 750, 500, 300, 150, and 50 bp, RNA dilutions: 10
-1
,
10
-2
, 10
-3
, 10
-4
, 10
-5
, and 10
-6
.
585
The target amplicon is usually smaller, reducing
the potential problems caused by target degradation.
Detection of specific gene sequences by the rRT-PCR
involves monitoring of the fluorescence generated by
cleavage of a target specific oligonucleotide probe
during amplification. This eliminates the need to open
the reaction tube post-amplification, neither for a nested
step, nor final agarose gel analysis of the cDNA
products, greatly reducing the risk of cross-
contamination.
The real time assay using the TaqMan Probe
has been developed to identify RHDV infection in
convalescent rabbits (8). The ability of one-step
multiplex TaqMan rRT-PCR to detect viral RNA was
measured at 10 copies/well. According to these data, a
linearity over a range from 10
1
to 10
10
copies was
demonstrated.
This study describes a highly sensitive rRT-
PCR in samples of the liver taken from RHDV infected
rabbits. It was confirmed that all liver specimens
originated from naturally or experimentally infected
rabbits, recognised as positive by other virological tests,
were positive in the real time RT-PCR with mean Ct
values close to 21. Of 26 specimens, four were positive
at higher Ct values, but always below 35 cycles. It was
shown that the limit of detection of viral RNA extracted
from the liver of rabbits infected with known virus
strains was established between 10
-7
and 10
-8
dilution.
Additionally, using real time RT-PCR test it was found
that the test is more sensitive than conventional RT-PCR
test, since RNA of KGM strain was detected at a 10
-5
dilution (Figs 1, 2).
The examination of liver homogenates of
apparently healthy rabbits revealed the presence of viral
RNA in two rabbits, as it was detected by both rRT-PCR
and RT-PCR. In three samples viral RNA was not
detected and the remaining samples slightly exceeded
the Ct value of the threshold. Of 13 serum samples taken
from seropositive rabbits, which have been in contact
with RHD virus, only two revealed Ct value in the
established limit.
In conclusion, the applied one-step TaqMan
rRT-PCR method is a sensitive and reliable technique
for RHDV detection. This method is more sensitive and
much faster to perform than the conventional RT-PCR. It
was confirmed that the specific oligonucleotide primers,
TaqMan probe, and the used reagent kit can detect the
genetic material of RHD virus in the liver of infected
rabbits, in a wide range of concentration of viral
particles. The test can be included as a new diagnostic
tool in RHD diagnosis.
References
1. Anon.: OIE, World Organisation for Animal Health –
OIE Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals, sixth edition, chapter 2.6.2., 2008,
pp. 947-961.
2. Cancellotti F.M., Renzi M.: Epidemiology and current
situation of viral haemorrhagic disease of rabbits and the
European brown hare syndrome in Italy. Rev Sci Tech
Off Int Epiz 1991, 10, 409-422.
3. Capucci L., Fusi P., Lavazza A., Pacciarini M.L., Rossi
C.: Detection and preliminary characterization of a new
rabbit calicivirus related to rabbit hemorrhagic disease
virus but nonpathogenic. J Virol 1996, 70, 8614-8623.
4. Capucci L., Fallacara F., Grazioli S., Lavazza A.,
Pacciarini M.L., Brocchi E.: A further step in the
evolution of rabbit hemorrhagic disease virus: the
appearance of the first antigenic variant. Virus Res 1998,
58, 115-126.
5. Chasey D., Lucas M. H., Westcott D. G., Sharp G.,
Kitching A., Hughes S. K.: Development of diagnostic
approach to the identification of rabbit haemorrhagic
disease. Vet Rec 1995, 137, 158-160.
6. Fitzner A.: Viral haemorrhagic disease virus of rabbits
and its immunogenic properties. PhD Thesis. NVRI,
Pulawy, Poland, 1994.
7. Fitzner A., Kęsy A., Niedbalski W., Paprocka G.:
Application of RT-PCR for identification of viral
haemorrhagic disease virus (RHDV) of rabbits isolated
in Poland. Medycyna Wet 1999, 55, 120-122.
8. Gall A., Hoffmann B., Teifke J.P., Lange B., Schirrmeier
H.: Persistence of viral RNA in rabbits which overcome
an experimental RHDV infection detected by a highly
sensitive multiplex real-time RT-PCR. Vet Microbiol
2007, 120, 17-32.
9. Górski J., Mizak B., Mizak Z., Komorowski A.: Clinical
and anatomopathological features of rabbit peste (viral
haemorrhagic disease of rabbits). Życie Wet 1988, 63,
266-269.
10. Kęsy A., Fitzner A., Niedbalski W., Paprocka G.,
Walkowiak B.: A new variant of the viral haemorrhagic
disease of rabbits virus. Rev Sci Tech Off Int Epiz 1996,
15, 1029-1035.
11. Goud A.R., Kattenbelt J.A., Lenghaus C., Morissy C.,
Chamberlain T., Collins B.J., Westbury H.A.: The
complete nucleotide sequence of rabbit haemorrhagic
disease virus (Czech strain V351): use of the polymerase
chain reaction to detect replication in Australian
vertebrates and analysis of viral population sequence
variation. Virus Res 1997, 47, 7-17.
12. Gregg D.A., House C., Meyer R., Berninger M.: The
viral haemorrhagic disease of rabbits in Mexico:
epidemiology and viral characterization. Rev Sci Tech
Off Int Epiz 1991, 10, 435-451.
13. Guittré C., Baginski I., Le Gall G., Trépo C., Cova L.:
Detection of rabbit haemorrhagic disease virus isolates
and sequence comparison of the N-terminus of the
capsid protein gene by the polymerase chain reaction.
Res Vet Sci 1995, 58, 128-132.
14. Guittré C., Ruvoen-Clouet N., Barraud L., Cherel Y.,
Baginski I., Prave M., Ganiere J.P., Trépo C., Cova L.:
Early stages of rabbit haemorrhagic disease virus
infection monitored by polymerase chain reaction. J Vet
Med 1996, B 43, 109-118.
15. Lancioti R.S., Kerst A. J.: Nucleic acid sequence-based
amplification assays for rapid detection of West Nile and
St. Louis encephalitis viruses. J Clin Microbiol 2001, 39,
4506-4515.
16. Liu S.J., Xue H.P., Pu B.Q., Quian N.H.: A new viral
disease in rabbits. Anim Husb Vet Med 1984, 16, 253-
255.
17. Meyers G., Wirblich C., Thiel H-J.: Rabbit hemorrhagic
disease virus – molecular cloning and nucleotide
sequencing of a calicivirus genome. Virology 1991, 184,
664-676.
586
18. Morisse J-P, Le Gall G., Boilletot E: Hépatites d’origine
virale des Léporidés: introduction et hypothèses
étiologiques. Rev Sci Tech Off Int Epiz 1991, 10, 269-
282.
19. Ohlinger V.F., Haas B., Thiel H.J.: Rabbit hemorrhagic
disease (RHD): characterization of the causative
calicivirus. Vet Res 1993, 24, 103-116.
20. Parra F., Prieto M.: Purification and characterization of a
calicivirus as the causative agent of a lethal hemorrhagic
disease in rabbits. J Virol 1990, 64, 4013-4015.
21. Schirrmeier H., Reimann I., Kollner B., Granzow H.:
Pathogenic, antigenic and molecular properties of rabbit
haemorrhagic disease virus (RHDV) isolated from vac-
cinated rabbits: detection and characterization of anti-
genic variants. Arch Virol 1999, 144, 719-735.
