Real-time fluorogenic reverse transcription polymerase chain

452723
trago et al.Real-time RT-PCR for Bagaza virus
JVDXXX10.1177/1040638712452723Bui
Real-time fluorogenic reverse transcription
polymerase chain reaction assay for the
specific detection of Bagaza virus
Journal of Veterinary Diagnostic Investigation
24(5) 959­–963
© 2012 The Author(s)
Reprints and permission:
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DOI: 10.1177/1040638712452723
http://jvdi.sagepub.com
Dolores Buitrago, Ana Rocha, Cristina Tena-Tomás, Marta Vigo,
Montserrat Agüero, Miguel Angel Jiménez-Clavero1
Abstract. In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ringnecked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological
agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in
India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods
to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase
chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers
targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely
related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated
using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges
during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RTPCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The
results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid
technique for BAGV genome detection, useful in both diagnosis and surveillance studies.
Key words: Bagaza virus; diagnostics; Flavivirus; real time; reverse transcription polymerase chain reaction.
Viruses belonging to the Flavivirus genus, such as Japanese
encephalitis virus, West Nile virus (WNV), Usutu virus, and
Zika virus, have repeatedly demonstrated the ability to cause
infection beyond what has traditionally been considered their
geographical range, as previously reviewed.17 Bagaza virus
(BAGV) is a member of the Flavivirus genus (family Flaviviridae), belonging to the Ntaya group.11 BAGV has become
another example of the spread of a flavivirus to a new continent, which in this case is Europe. Before 2010, BAGV had
only been identified in mosquitoes in Central and Western
Africa6,7,16 and in India.3 In all cases, it was found in mosquitoes in the context of surveillance activities addressed to other
viruses causing diseases of relevance, both for animals and
human beings. In India, serological studies suggested that it
might be able to infect human beings,3 although such pathogenicity of the virus is currently unknown. Sequence homology suggested that BAGV is synonymous to Israel turkey
meningoencephalomyelitis virus (TMEV),12 a pathogen
affecting turkeys that was first described in Israel in 195810
and which has been repeatedly isolated in that country.4 Outside Israel, TMEV has only been reported in South Africa.2
In September 2010, BAGV was detected and identified in
the southernmost province of Spain (Cádiz), as the etiological agent of an outbreak in wild birds.1 The outbreak caused
mortality in 2 bird species (red-legged partridges, Alectoris
rufa; ring-necked pheasants, Phasianus colchicus), which
were found dead in different hunting properties. As valued
wild game birds, both species are often reared in captivity
and released for hunting purposes. The red-legged partridge
is native to Spain, where wild populations are still abundant,15
whereas the ring-necked pheasant (or, common pheasant) is
an exotic, introduced game species.5
BAGV was identified as the causative agent of the outbreak during laboratory investigations using a pan-flaviviral
hemi-nested reverse transcription polymerase chain reaction
(RT-PCR) assay, amplifying a fragment of the nonstructural
NS5 protein coding region of the viral RNA,14 which was
subsequently sequenced to confirm the identity of the viral
genome detected.1 However, this method is cumbersome,
time consuming, and, as often happens with nested PCR
methods,9 prone to cross-contamination, and therefore is not
suitable for efficient diagnosis and disease surveillance. With
the aim to improve the efficacy and speed of the diagnosis of
From the Laboratorio Central de Veterinaria, Algete, Spain (Buitrago,
Rocha, Tena-Tomás, Vigo, Agüero), and CISA-INIA, Valdeolmos, Spain
(Jiménez-Clavero).
1
Corresponding Author: Miguel Angel Jiménez-Clavero, CISA-INIA,
Ctra Algete-ElCasar, s/n, 28130, Valdeolmos (Madrid) Spain.
[email protected]
960
Buitrago et al.
Figure 1. Sequence alignment showing the position of the primers and probe designed in the current study with regard to the nucleotide
sequence of Bagaza virus (BAGV) and of other related flaviviruses (names of strains on the left: see text for more details). The genome
stretch represented in the figure corresponds to a part of the NS5 gene of the related flaviviruses (nucleotide positions 8977–9048 according
to GenBank accession no. EU684972).
this disease, and to enable surveillance based on the detection of the BAGV genome, a real-time RT-PCR method specific for this virus was developed, and its diagnostic
performance was evaluated using field samples and virus
isolates from the first outbreak of this disease in Spain. The
present article describes the development and evaluation of
this new technique.
As a first step, nucleotide primers and a commercial probe
aimed at the specific detection of BAGV were designed based
on multiple alignments comprising a highly conserved
sequence within the NS5 region of different flaviviruses. The
alignment included sequences from 3 different BAGV strains
(African strain Dak Ar B209, Indian strain 96363, and Spanish
strain H/2010), 4 different WNV strains (Kunjin strain
MRM61C, NY99, PT6.39, and Eg101), 1 Usutu virus strain
(SAAR-1776), and 1 Zika virus strain (MR 766; GenBank
accession nos. AY632545, EU684972, HQ644144, D00246,
DQ211652, AJ965630, EU081844, AY453412, and AY632535,
respectively). Sequences were aligned using commercial
software.a Based on this alignment (Fig. 1), a primer pair and a
probe specific for BAGV were designed using commercial
software.b The forward primer (nucleotide positions 8980–
8999, numbered according to GenBank EU684972) was
5’-GGAAGCAGGGCCATATGGTA-3’, the reverse primer
(nucleotide positions 9041–9020, numbered as above) was
5’-CGAGGGCCTCAAAYTCTARRAA-3’, and the probe
(nucleotide positions 9005–9017, numbered as above) consisted of 5’-FAM-TGGCTYGGATCCC-MGB-3’. The probe
was labeled with 6-carboxyfluorescein (FAM) and at the 3’-end
with a nonfluorescent quencher bound to an minor groove
binder (MGB) group.c
The probe and 1 of the selected primers contained, respectively, 1 and 3 degenerate positions to improve the specificity of the real-time RT-PCR for BAGV (Fig. 1). To achieve
the best conditions for RT-PCR amplification, the protocol
was optimized by testing different concentrations of primers
and probe. The final protocol consisted of a 1-step RT-PCR
with the following mixture: RT-PCR buffer,d RT-PCR
enzyme mix,d forward primer (0.7 µM final concentration),
reverse primer (0.7 µM final concentration), probe (0.15 µM
final concentration), and 3 µl of template in a total of 20 µl of
reaction volume. Amplification and fluorescence detection
were conducted in real-time PCR equipmente using a program consisting of a reverse transcription step at 48ºC for
25 min followed by inactivation and denaturation at 95ºC
for 10 min, and a PCR amplification cycle of 40 cycles of
95ºC for 15 sec, 52ºC for 30 sec, and 60ºC for 30 sec.
Fluorescence data were acquired at the end of the 60ºC step.
A hemi-nested RT-PCR specific for a broad range of flaviviruses14 (the same used in the diagnosis of the outbreak) was
performed in parallel in all the samples analyzed in this
work, to allow a comparison.
The sensitivity of the new method was analyzed by testing either serial dilutions of viral RNA or viral suspensions
(extracted using an automated procedure,f according to the
manufacturer’s protocol) from 200-µl aliquots of cell-culture
propagated virus seeds (titrated in median tissue culture
infectious doses [TCID50]/ml by a standard limiting dilution
titration assay13 using BSR cells, virus titer: 104 TCID50/ml)
of a cell culture-grown BAGV isolate Spain H/2010 from the
heart of an affected partridge in the 2010 outbreak in Spain.1
As a result, the real-time RT-PCR method for BAGV genome
detection showed a detection limit of 10-1 TCID50/ml,
which was comparable to the sensitivity achieved by the
pan-flaviviral hemi-nested RT-PCR method performed in
parallel (Fig. 2).
To study the specificity of the BAGV real-time RT-PCR,
purified RNA samples from different flaviviruses, including
BAGV isolate Spain H/2010, as well as non-BAGV flaviviruses (Usutu virus strain SAAR-1776; Japanese encephalitis
virus strain Nakayama; West Nile virus strains NY99,
PT6.39, Eg101, and Kunjin MRM 16), and other nonflaviviral avian viruses, including avian Influenza A virus (H5N2,
H5N3, H7N2, H7N9), Newcastle disease virus (mesogenic and
lentogenic strains), Beak and feather disease virus, and Gallid
herpesvirus 2, were analyzed. The new real-time RT-PCR
gave positive results only with BAGV-derived RNA, but not
with RNA from other flaviviruses (whose integrity was confirmed using the broad range of pan-flaviviral hemi-nested
RT-PCR; data not shown) or from other avian viruses (whose
Real-time RT-PCR for Bagaza virus
961
Figure 2. Comparison of sensitivity for Bagaza virus (BAGV) using 2 methods performed in parallel: A, gel-based pan-flaviviral reverse
transcription polymerase chain reaction (RT-PCR; above) and hemi-nested RT-PCR (below), and B, real-time RT-PCR specific for BAGV.
Serial 10-fold dilutions of BAGV cell-culture supernatant (virus titer: 104 TCID50/ml) were prepared (≤10-8) and analyzed by the 2 methods
in parallel. In panel A, lanes 1–7 correspond to 10,000, 1,000, 100, 10, 1, 0.1, and 0.01 median tissue culture infective doses (TCID)50/
ml, respectively, whereas lane 8 is a control test. In panel B, the results obtained with the same serial virus dilutions as in A, with the
exception of the undiluted sample, and expressed as Log TCID50/ml, are represented versus threshold cycle (Ct) obtained in each real-time
RT-PCR reaction, performed by duplicate. The regression line is represented, and regression equation, square of correlation coefficient, and
efficiency obtained are included in the plot.
integrity was confirmed with PCR or RT-PCR methods specific for each virus; data not shown). This result confirms the
specificity of the real-time RT-PCR method for BAGV.
To evaluate the performance of the newly developed realtime RT-PCR method in clinical samples, specimens were
obtained from red-legged partridges (n = 11) from the disease outbreak that occurred in Cádiz in 2010. The samples
were originally submitted for diagnosis to the authors’ laboratory at the National Reference Laboratory for Arboviral
Diseases of Animals (Spain). Samples consisted of different
tissues and/or organs, including heart, intestine, lung, liver,
kidney, brain, and feathers from affected as well as nonaffected partridges, which were conserved frozen at –70ºC
until used in this work. The samples were previously analyzed by real-time RT-PCR specific for lineage 1 and lineage
2 WNV,8 all giving negative results in this test, and by the
pan-flaviviral hemi-nested RT-PCR. Tissues and/or organs
were homogenized in a volume of 200 µl in phosphate buffered saline using commercial equipment,g following the
manufacturer’s instructions, and the homogenates obtained
were subjected to nucleic acid extraction as above. As a
result, all samples identified as positive by hemi-nested
RT-PCR were also positive with the new real-time RT-PCR
developed in the present study, confirming that the diagnostic
sensitivity of real-time RT-PCR is equivalent to the heminested RT-PCR (Table 1). Also, samples from partridges collected during surveillance studies in 2010 and 2011 (n = 81)
that yielded negative results in the pan-flaviviral hemi-nested
RT-PCR were also negative in the new real-time RT-PCR for
BAGV (data not shown).
In summary, a real-time RT-PCR method was developed
that was specifically designed to detect the presence of
BAGV genome in clinical samples with a high sensitivity.
This new method proved to be valid for the detection of
BAGV genome either in virus isolates or in clinical samples
from affected animals, providing a sensitivity equivalent to
the gel-based pan-flaviviral RT-PCR method previously
employed. As compared to the gel-based RT-PCR, the new
method offers several advantages, besides its specificity to
BAGV. The new method reduces the time required for analysis (less than 4 hr following receipt of the samples) without
any loss of sensitivity or specificity. The assay also significantly reduces the risk of false-positive results due to crosscontamination, which is a well-known drawback of nested
PCR protocols.9 In addition, in combination with an automated extraction system, this new method would enable
large-scale analysis of the virus in animal populations potentially affected with the disease, a feature that may be useful
962
Buitrago et al.
Table 1. Comparative analysis of clinical samples from affected red-legged partridges (Alectoris rufa) in the Bagaza virus (BAGV)
outbreak in 2010 using a newly developed real-time reverse transcription polymerase chain reaction (RT-PCR) specific for BAGV and a
pan-flaviviral hemi-nested RT-PCR method.*
No. positive/no. tested
Tissue/sample type
No. of animals analyzed
BAGV-specific real-time RT-PCR
Pan-flaviviral hemi-nested RT-PCR
10
1
1
1
1
1
1
1
1
1
10/10 (18.6–28.8)
1/1 (27.7)
1/1 (33.5)
1/1 (33.4)
1/1 (24.0)
1/1 (25.0)
1/1 (22.6)
1/1 (27.1)
1/1 (20.5)
1/2 (33.0)
10/10
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/2
Brain
Cloacal swab
Oral swab
Gut
Heart
Kidney
Lung
Liver
Feathers
*Threshold cycle value in parentheses.
in case of recurrence in the area and/or spread to other areas.
This method provides the diagnostic laboratory with an
effective and rapid analytical tool, useful in differential diagnosis and surveillance of this flaviviral disease of birds,
which could represent an emerging animal health risk in
Europe.
Sources and manufacturers
a. ClustalW software (http://www.ebi.ac.uk/Tools/clustalw/), European
Bioinformatic Institute, Cambridge, UK.
b. Primer Express (version 2.0.0) software, Applied Biosystems,
Branchburg, NJ.
c. TaqMan–MGB Probe, Applied Biosystems, Branchburg, NJ.
d. AGPATH-ID ONE RT-PCR kit, Applied Biosystems,
Branchburg, NJ.
e.7500 Fast Real Time PCR System, Applied Biosystems,
Branchburg, NJ.
f. BioSprint DNA Blood Kit, BioSprint 96 Workstation; Qiagen
Inc., Valencia, CA.
g. MagNA Lyser, Roche Diagnostics, Indianapolis, IN.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
study has been funded by the Ministries of Agriculture, Food and
Environment, and Economy and Competitiveness of Spain (grant
AGL2011-13634-E).
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