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Detection of RHD virus by a real-time reverse transcription PCR

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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.
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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.
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... Some inconvenience of this system is the fact that this fluorochrome non-specifically binds to double-stranded DNA, yet in order to exclude the possibility of wrong interpretation, the analysis of product melting curves must be performed (Kubista et al. 2006, Watzinger et al. 2006, Lipiński et al. 2008). Real-time PCR method, as an element of viral diagnostics, was applied to assessment of viruses of DNA from the Parvoviridae (Dzieciątkowski et al. 2007), Herpesviridae (Grabarczyk et al. 2003, Radkowski et al. 2005, Dzieciątkowski et al. 2008, Poxviridae (Bacławski (Bae et al. 2003, Pejsak et al. 2006, Gurukumar et al. 2009), Retroviridae (Kuźmiak et al. 2006) and Caliciviridae (Nowaczyk 2007, Fitzner et al. 2011, Teixeira et al. 2011 families. Within the last family, it was used for assessment of the volume of viral copies in the peripheral blood of rabbits infected with EBHS (European Brown Hare Syndrome), which is a pathogenic factor for hares (Nowaczyk 2007). ...
... Research performed by Teixeira et al. (2011), referred to the use of a new method for isolation of the RHD virus from liver tissue, namely centrifugation at high rpm in iodixanol gradient, which can be very useful for further "processing", e.g. using real-time PCR. Also Fitzner et al. (2011) applied the real-time PCR method for assess-ment of 26 strains of the RHD virus (KGM 1988, SGM 1988, PD 1989, BLA 1994, MAL 1994, PRB 1995, BDG 1/1996, BDG 2/1997, BDG 3/1997, BDG 4/1998, GSK 1998, PIA 1999, POZ 199, ZD0 2000, SIZ 202, ZDU 2003, OPO 2004, GRZ 2004, ROK 2004, CB 2005, KRY 2005, ZKA 2005, DCE 2006, in which not only the presence of RHD alone was assessed in particular liver samples. It was also shown that the limit of detection of viral RNA extracted from livers is at the 10 -7 dilution. ...
... Thus the optimalisation and validation of the method was appropriate as it confirms the clinical observations (mortality of infected animals). Firstly, the conservative region primers, which were also applied in the study by Fitzner et al. (2011), permitted detection of all analysed strains of the RHD virus, and the result was not affected by differences recorded among the seventeen analysed strains of the RHD virus as regards their biological properties, such as haemagglutination capacity or formation of antigen variants, and time and place of isolation of such strains. Furthermore, various pathogenicity, measured using the mortality percentage of rabbits infected with the analysed strains, also did not affect the positive result of the real-time PCR reaction, which points to the fact that the method is unusually sensitive, and is not dependent on the volume of material to be detected, in this case the number of viral particles in the liver. ...
Article
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The paper concerns the use of a novel, very effective diagnostic method, a real-time PCR for diagnosis of a viral agent causing viral haemorrhagic disease in rabbits - RHDV. Until now, the method was widely used for detecting many different viruses, both DNA, and RNA, but as far as RHDV is concerned, there are not many records of such use. This study aimed at the detection of 17 different strains from different European regions, differing in biological features and mortality. The study confirmed that real-time PCR is an applicable and effective method for diagnosis of RHDV, irrespective of the stains' features.
... A comparison between real-time RT-PCR and conventional RT-PCR highlights some of its advantages, namely the reduced risk of cross-contamination. To this greatly contributes the use of a one-tube protocol that avoids post-PCR handling, which in turn significantly shortens the reaction and the diagnostic times [7,156,158,159]. RNA virus degradation is less challenging as the target amplicons are usually smaller, thus also increasing sen-sitivity [7,158], and intra and inter-assay variability is low [154,156]. ...
... To this greatly contributes the use of a one-tube protocol that avoids post-PCR handling, which in turn significantly shortens the reaction and the diagnostic times [7,156,158,159]. RNA virus degradation is less challenging as the target amplicons are usually smaller, thus also increasing sen-sitivity [7,158], and intra and inter-assay variability is low [154,156]. Detection limits of RHDV real-time RT-PCRs range from 9-100 copies [7,27,156,160] and have been shown to be similar to those of nested PCR [155]. ...
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Since the early 1980s, the European rabbit (Oryctolagus cuniculus) has been threatened by the rabbit hemorrhagic disease (RHD). The disease is caused by a lagovirus of the family Caliciviridae, the rabbit hemorrhagic disease virus (RHDV). The need for detection, identification and further characterization of RHDV led to the development of several diagnostic tests. Owing to the lack of an appropriate cell culture system for in vitro propagation of the virus, much of the methods involved in these tests contributed to our current knowledge on RHD and RHDV and to the development of vaccines to contain the disease. Here, we provide a comprehensive review of the RHDV diagnostic tests used since the first RHD outbreak and that include molecular, histological and serological techniques, ranging from simpler tests initially used, such as the hemagglutination test, to the more recent and sophisticated high-throughput sequencing, along with an overview of their potential and their limitations.
... This technique is known to be more efficient than conventional PCR in the detection and quantification of small numbers of viral particles. 59,111 It has been employed successfully for detection of RHDV strains circulating in rabbit populations in various European countries 1,16,22,30,35,38,86,87 and Australia 77 or for studying virus distribution in tissues of experimentally infected rabbits. 71 Broadly reactive primers within the conserved 3'-region of the VP60 RHDV gene were designed and used in the Taq-Man 35 and SYBR Green 71,86 RT-rtPCR methods. ...
... 71 Broadly reactive primers within the conserved 3'-region of the VP60 RHDV gene were designed and used in the Taq-Man 35 and SYBR Green 71,86 RT-rtPCR methods. When compared with conventional RT-PCR, RT-rtPCR was 100 times more sensitive, 35 with its detection level established at 6.09 genome copies per reaction. 71 The emergence of new RHDV-2 strains in rabbits initiated research toward the development of strain-specific TaqMan RT-rtPCR methods. ...
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Various PCR-based assays for rabbit viruses have gradually replaced traditional virologic assays, such as virus isolation, because they offer high-throughput analysis, better test sensitivity and specificity, and allow vaccine and wild-type virus strains to be fully typed and differentiated. In addition, PCR is irreplaceable in the detection of uncultivable or fastidious rabbit pathogens or those occurring in low quantity in a tested sample. We provide herein an overview of the current state of the art in the molecular detection of lagomorph viral pathogens along with details of their targeted gene or nucleic acid sequence and recommendations for their application. Apart from the nucleic acids–based methods used for identification and comprehensive typing of rabbit viruses, novel methods such as microarray, next-generation sequencing, and mass spectrometry (MALDI-TOF MS) could also be employed given that they offer greater throughput in sample screening for viral pathogens. Molecular methods should be provided with an appropriate set of controls, including an internal amplification control, to confirm the validity of the results obtained.
... The analysis of the results showed that real-time PCR is a highly efficient and effective method for diagnosing L. europaeus GI.1 infections [36]. Fitzner et al. [37] decided to perform a comparative analysis of the results obtained by conventional PCR and real-time PCR in the diagnosis of infection in the livers of rabbits experimentally infected with 26 strains of L. europaeus GI.1. Analysis of the obtained results revealed linearity ranging from 10 1 to 10 10 copies, which was consistent with the expected values. ...
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Featured Application Our experience with real-time PCR, together with several different studies in this field show that this is a very useful method for rapid, specific and effective virus detection. Abstract Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.
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The causative viral agent of a lethal rabbit hemorrhagic disease has been purified and characterized. In negative-stained preparations, the virions were icosahedral, measured 27 to 35 nm in diameter, were without an envelope, and showed 10 peripheral cup-shaped depressions. The major structural protein was 60 kilodaltons, which constitutes a unique characteristic of the Caliciviridae.
Article
The aim of this study was to apply the method of reverse transcription coupled with PCR for the detection of RHDV RNA in livers of infected rabbits. All tested RHDV strains were isolated from outbreaks occuring in Poland over the last 10 years. The viral RNA was extracted by the guanidine thiocyanate-phenol-chloroform method or using the Rneasy Mini Kit (Qiagen) for rapid isolation of RNA. Each sample of purified RNA was reversly transcribed using AMV reverse transcriptase and antisense P5 (7071) primer chosen from 3′-end of the viral genome. The amplification of cDNA was performed in a reaction mixture containing two different pairs of primers: P1, P2 (320 bp) and P5, P6 (510 bp) frond the capsid coding region of the viral genome. All isolates of RHDV gave positive RT-PCR products. RNA from livers of uninfected healthy rabbits gave negative results. The PCR technique is a rapid, specific and accurate diagnostic method and it can be useful for the detection of RHDV in diagnostic materials.
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Rabbit hemorrhagic disease virus (RHDV) is a noncultivable calicivirus that infects rabbits (Oryctolagus cuniculus) and causes epidemics of an acute fatal hepatitis. In 1997 we identified two RHDV isolates from geographically distant Italian regions, which differed antigenically from the reference strain RHDV.Bs89. In fact, they were not reactive with mAb 1H8, that is able to protect rabbits from RHD and showed a low reactivity with the rabbit convalescent serum raised against RHDV.Bs89. Experimental infection of rabbits with either RHDV isolates confirmed their high pathogenicity and their peculiar antigenic profile; nevertheless, rabbits vaccinated with the current vaccine were protected against challenge infection with these isolates. Sequence comparison definitely demonstrated that the two isolates originated from the same RHDV variant and that the similarity of their structural protein (VP60) sequences with the RHDV.Bs89 is equal to 93%. This variant was named RHDVa since shows consistent genetic and antigenic differences from the wild-type RHDV. In particular, 44% of amino acid substitutions in RHDVa VP60 were located between amino acids 344 and 370, where the similarity with RHDV.Bs89 drops to 70%, suggesting that this region probably contains the epitope recognized by mAb 1H8. In addition, this paper presents preliminary data concerning the amino acids of VP60 involved in the hemagglutination site of the virus.
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Rabbit production is of considerable economic importance in Italy. In the last thirty years, meat production has risen and the number of intensive husbandry establishments has grown. The major region of production (about 60%) lies in the northern part of the country. In addition, approximately one million live animals and more than 14,000 tons of meat are imported yearly. More than 150,000 hares are also imported for restocking and hunting purposes. During the second half of the 1980s a "new" disease (X disease), characterised by haemorrhagic and degenerative lesions and high mortality, was observed on traditional farms in some regions. This form spread throughout the country between 1986 and 1988. In 1988, diagnostic research led to identification of a viral agent. Because of its morphology, the virus was at first considered a picornavirus. More recent research has included the virus in the Caliciviridae family. This agent reproduces the disease experimentally. Between 1988 and 1989, many intensive establishments in several regions were affected by the disease, known as viral haemorrhagic disease (VHD); the losses were always very high. In 1989, after mortality among free-living hares had been observed in the northern part of Italy for some years, the agent of the European brown hare syndrome (EBHS) was identified as being morphologically similar to the VHD virus. The antigenic relationship between VHD and EBHS and the possibility of cross-infection between rabbits and hares are currently being studied. Hygienic measures and vaccination are the most reliable methods of control. International cooperation has greatly helped in the study of the disease and in the adoption of control measures.
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A fatal disease of rabbits was first reported in the People's Republic of China in 1984. Since 1986, the disease has been reported in most countries of Europe and in the Republic of Korea. In 1989 a similar disease, presumably linked to the importation of rabbit meat from the People's Republic of China, spread rapidly through ten states in Mexico; it was eradicated during the same year by "stamping-out" measures. In Mexico, as was the case in other outbreaks, morbidity and mortality reached 80-90% with few clinical signs. In pathogenesis studies, the primary sites of replication were in the small intestinal crypt and villous epithelium, hepatocytes and splenic lymphocytes. Many organs, including the lung and kidney, contained acutely infarcted tissue and haemorrhages resulting from a terminal disseminated intravascular coagulopathy. The disease and the characteristics of the virus isolated in Mexico are similar to isolates from Europe and the Republic of Korea. The comparative morphologic, from Europe and the Republic of Korea. The comparative morphologic, immunologic, and in situ nucleic acid hybridization evidence for a parvovirus aetiology are summarized.
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The RNA genome of rabbit hemorrhagic disease virus (RHDV) was molecularly cloned. The 5' terminal sequence of the genomic RNA was determined after PCR amplification of a G-tailed first strand cDNA template. The cloned cDNA allowed determination of the first complete caliciviral sequence encompassing 7437 nucleotides without poly(A) tail. The RHDV genome contains one long open reading frame of 2344 codons which in the 5' region encodes the nonstructural proteins. Sequence comparison studies revealed significant homology between nonstructural proteins of the feline calicivirus (FCV) and RHDV. In analogy to FCV the deduced RHDV amino acid sequence contains a picornavirus 2C-like sequence, a hypothesized cysteine protease motif, and the conserved polymerase residues GDD. For the protein region containing the GDD motif, alignments of sequences from different viruses including the putative caliciviruses hepatitis E virus and Norwalk virus were performed; concerning the classification of the latter two viruses, a final judgement was not possible. Bacterial expression of sequences derived from the 3' part of the genomic RHDV RNA showed that this region codes for the viral capsid protein.
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At present there is no sensitive method for the detection of rabbit haemorrhagic disease virus (RHDV), a calicivirus causing high mortality in rabbit populations. For this purpose a reverse transcriptase polymerase chain reaction (RT-PCR) was established in the N-terminal portion of the RHDV capsid region. The RT-PCR was 10(4)-fold more sensitive than ELISA testing for the detection of the virus and was able to detect as few as 12 copies of template cDNA. By using the RT-PCR test and sequencing, 96.6 to 98.7 per cent homology was demonstrated in the N-terminal portion of the capsid protein of three isolates from geographically and temporally separate outbreaks of viral haemorrhagic disease, indicating that this portion of the RHDV capsid protein is highly conserved.
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Rabbit hemorrhagic disease (RHD) which was first recognized in China in 1984 spread via Eastern Europe to many countries of Western Europe and other parts of the world. The analysis of the virus outlined in this review comprises: 1) physico-chemical properties, 2) electron microscopy including immunoelectron microscopy, 3) demonstration of capsid protein, 4) in vivo neutralization with monoclonal antibodies (mabs), 5) infectivity of purified RNA, and 6) characterization of the viral genome. Also included are clinical, pathological and epidemiological findings, different diagnostic methods as well as disease control measures. Finally, similarities between RHD and the European brown hare syndrome (EBHS) are pointed out. The latter disease is caused by a calicivirus different from RHDV.
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Haemagglutination and ELISA tests, and negative contrast electron microscopy, have been used to identify rabbit haemorrhagic disease virus in naturally occurring cases of the disease and in experimentally infected rabbits in the United Kingdom. Haemagglutination tests alone are not satisfactory for the diagnosis because non-haemagglutinating isolates of the virus, otherwise indistinguishable from others, have been found in some outbreaks. Haemagglutination inhibition tests have shown that a proportion of both commercial laboratory and wild rabbits in the UK are seropositive to the virus although they have not been associated with clinical disease. This observation, made previously in other parts of Europe, may indicate the longstanding circulation of a related but non-pathogenic strain of virus. Naturally occurring antibody appears to afford a high degree of protection against experimental challenge with virulent virus.