Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.Centro de Estudios Parasitológicos y de Vectore

Agirre, Jon

Guérin, Diego M. A.

Martí, Gerardo Aníbal

Querido, Jailson F. B.

Silva, Marcelo Sousa

BioMed Research International

SEDICI - Repositorio de la UNLP

Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 218593, 3 pages
http://dx.doi.org/10.1155/2013/218593
Methodology Report
Molecular Techniques for Dicistrovirus Detection without
RNA Extraction or Purification
Jailson F. B. Querido,1 Jon Agirre,2 Gerardo A. Marti,3
Diego M. A. Guérin,2 and Marcelo Sousa Silva1
1 Centre for Malaria and Tropical Diseases, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Rua da Junqueira, 100, 1349-008 Lisboa, Portugal
2 Unidad de Biof´ısica (UBF), CSIC-UPV/EHU, Barrio Sarriena S/N, Bizkaia, 48940 Leioa, Spain
3 Centro de Estudios Parasitolo´gicos y de Vectores (CEPAVE), CCT-La Plata/CONICET-UNLP, No. 584, 1900 La Plata, Argentina
Correspondence should be addressed to Marcelo Sousa Silva; mssilva@ihmt.unl.pt
Received 10 January 2013; Revised 15 March 2013; Accepted 21 March 2013
Academic Editor: Zhirong Sun
Copyright © 2013 Jailson F. B. Querido et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect
pests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main
vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples
of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.
1. Introduction
Triatoma virus (TrV), together with Solenopsis invicta virus-
1, is one of the two members of the Dicistroviridae family
pathogenic to insects of medical importance [1]. Because of
its horizontal way of transmission and high pathogenicity
in populations of triatomines, TrV has been postulated to
be used as a biological control agent for both domiciliated
and nondomiciliated vectors of Chagas disease. It replicates
within the cytoplasm of gut cells of triatomines [2], causing a
high mortality rate, delayed development, and a reduction in
the fecundity [3, 4]. Recently, we have shown that this virus
is unable to replicate nor is infective in mice and probably is
not infective to other vertebrates, reinforcing the possibility
of this virus to be used as a tool in biological control of
triatomines transmitters of Chagas disease [5].
Dicistroviruses are characterized for having two open
reading frames, ORF1 andORF2.TheORF2 is responsible for
encoding the structural proteins VP1, VP2, VP3, and VP4,
which in the case of TrV have respective masses of 29.7 kDa,
28.4 kDa, 31.8 kDa, and 5.5 kDa [6]. The viral genome of TrV
is a +ssRNA molecule of 9010 nucleotides [6].
A reliable method for the detection of TrV by molec-
ular techniques represents an important step towards the
identification of insect species being affected by this virus
and the analysis of their geographical distribution as well.
However, the diagnosis of triatomines in regions where it is
difficult to conduct scientific investigation poses a serious
challenge. In this study, we present a new method that allows
the reduction of costs and time required for the entire process
of diagnosis, enabling the detection of TrV without resorting
to RNA extraction, andwithout running the risk of degrading
the RNA during the extraction, purification and storage
process.
2. Materials and Methods
2.1. Triatoma Virus Purification. The virus purification was
made according to [6, 7]; dry feces coming from infected
insects were collected on Whatman filter papers and excised
from them using a standard surgery scalpel until 2 g of
material was collected. A buffer composed of 10mMNaCl,
1mMMgCl
2
, and 200mM citric acid at pH 6 (Lysis buffer)
2 BioMed Research International
was added at a ratio of 100mL per gram of dry material.
PMSF (N 98.5%, purchased from Sigma) thawed at −20∘C
in isopropanol (Merck, GR for analysis) was added up to a
final concentration of 1mM. The mixture was homogenized
for 5min using a vortex mixer and then sonicated using
a Sanyo MSE Soniprep 150 operating at 10 s ON/10 s OFF
for 20 pulses. The homogenate was thereafter centrifuged in
a Kontron T-1170 ultracentrifuge for 45min at 20,000 gavg
using a Beckman SW28.1 rotor. The collected supernatant
was then centrifuged for 2.5 h at 180,000 gavg using a Kon-
tron TFT50.38 rotor. The resulting pellet was resuspended
overnight in 2mL of 10mMNaCl, 1mM MgCl
2
, and 50mM
Tris at pH 7.4 (NMTbuffer) and then loaded on top of a 35mL
continuous 5%–30% sucrose gradient.The centrifugationwas
at 100,000 gavg for 3 h in a Beckman SW28.1 rotor. The
gradient was fractionated from the bottom to the upper part
in 0.5mL aliquots that were collected in Greiner 96-well
MasterBlocks and sealed thereafter with a silicone-based lid.
The gradients were cooled in ice to avoid particle diffusion
within them. The individual optical density of each aliquot
was then measured at both 260 nm and 280 nm using an
Implen NanoPhotometer coupled with an LG100 UV-G6786
cell. A baseline correction was performed using equivalent
fractions from a control sucrose gradient. After plotting
the absorbance profile, selected aliquots were loaded onto
12.5% polyacrylamide SDS-PAGE gels to check the purity
of the samples. Following gradient fractionation, equivalent
samples were pooled together and dialyzed overnight against
2 L of NMT buffer.
2.2. Reverse Transcriptase-PCR. For the RT-PCR, we use
different concentration of purified TrV and feces samples
(66.7mg/mL), without resorting to RNA extraction. Dry
feces coming from infected insects were resuspended in a
buffer composed of 10mMNaCl, 1mMMgCl
2
, and 200mM
citric acid at pH 6 (Lysis buffer). PMSF (N 98.5%, purchased
from Sigma) thawed at −20∘C in isopropanol (Merck, GR
for analysis) was added up to a final concentration of 1mM.
The homogenate was centrifuged in an Eppendorf cen-
trifuge 5417 R for 30min at 20,000 gavg andwas subsequently
filtered through a 0.2 𝜇m. The feces sample was diluted in
DEPC-treated water to a final concentration of 33.3mg/mL.
As a positive control, we used viral RNA extracted (Tri-
zol, Invitrogen) from purified TrV and feces samples. The
cDNA synthesis was performed according to cDNA Synthe-
sis Kit Protocol (M-MuLV Reverse Transcriptase, Thermo
Scientific). In the PCR reactions were used single primers
pairs: TrV sense: 5󸀠-TCAAAACTAACTATCATTCTGG-3󸀠
(nt 7427 to 7448 in TrV ORF2 sequence) and TrV antisense:
5󸀠-TTCAGCCTTATTCCCCCCC-3󸀠 (nt 8240–8258 in TrV
ORF2 sequence), with an expected product of 832 bp [8],
selected from ORG2 region, conserved in dicistrovirus. PCR
products were visualized on 1.5% agarose gels (running
for 1 h at 100V) stained with ethidium bromide, and their
sizes were determined by comparison against DNA markers,
HyperLadder I (Bioline, UK).
M 1 2 3 4 5 6
10000
200
800
1000
(b
p)
Figure 1: PCR products on 1,5% agarose gel stained with ethidium
bromide. M: molecular weight marker 200 bp to 10000 bp (Bioline,
UK). Lane 1: negative control, lane 2: 3000 ng of purified TrV, lane 3:
300 ng of purified TrV, lane 4: 30 ng of purified TrV, lane 5: 3 ng of
purified TrV, and lane 6: 0,3 ng of purified TrV.
3. Results and Discussion
To obtain the cDNA, we used full genome TrV particles
without RNA extraction. We used a concentration range
between 3,000 and 0.3 ng of virus, being this concentration
measurement based on the viral proteins, diluted in DEPC-
treated water. The cDNA amplifications were performed
using the selected primers pairs mentioned above. We
obtained amplification until a minimum concentration of
3 ng of viral particles (Figure 1).
To initiate the cDNA synthesis, we mixed 5 𝜇L of viral
samples (5, 3, and 1 𝜇L for feces samples) with 100 pmol of
Oligo (dT), incubated first at 65∘C for 5min and then at
4∘C for 5min. During the contact with the host cells, the
virus released the genome. In vitro, this process may occur by
varying certain environmental conditions such as pH or tem-
perature [8, 9]. Incubating the virus at a temperature above
65∘C will cause RNA release [8]. However, such temperature
and time of incubation should not be too long because 65∘C is
close to the melting temperature of RNA.The results indicate
that viral RNA can be detected in feces samples, after a single
centrifugation and filtration step (Figure 2). These results
reinforce the applicability and reproducibility of the method
presented in the previous paragraph.
Whenwe compare the result of RT-PCR from the samples
analysed directly without resorting to RNA extraction to
the samples where RNA extraction was proceeded, we can
see that there is no significant difference between these two
methods, which indicates that the method proposed here is a
good alternative to the method traditionally used.
4. Conclusions
The method proposed in this study allows the detection
of dicistrovirus by RT-PCR without resorting to RNA
BioMed Research International 3
10000
1000
800
500
100
(b
p)
M 1 2 3 4 5 6 7 8 9 10 11
Figure 2: PCR products on 1,5% agarose gel. M: molecular weight
marker 100 bp to 10000 bp (Jena Bioscience, Germany). Lane 1:
negative control, lane 2: 5 𝜇L of viral RNA (from 3000 ng of purified
TrV), lane 3: 5 𝜇L of viral RNA (from 300 ng of purified TrV), lane 4:
5𝜇L of viral RNA (from 30 ng of purified TrV), lane 5: 5 𝜇L of viral
RNA (from 0,03 ng of purified TrV), lane 6: 5 𝜇L of viral RNA (from
1mg of feces), lane 7: 5 𝜇L of viral RNA (from 0.5mg of feces), lane
8: 5𝜇L of viral RNA (from 0,25mg of feces), lane 9: 5𝜇L of feces
samples (without RNA extraction), lane 10: 3𝜇L of feces samples
(without RNA extraction), and lane 11: 1 𝜇L of feces samples (without
RNA extraction).
extraction, which reduces the cost associated with this
method and the risk of RNA degradation during the process
of RNA extraction, and it is swimmingly reproducible in a
low-budget laboratory. Moreover, this study provides new
facts about the thermostability of the TrV structure.
Acknowledgments
Theauthors are grateful to Rube´n Sa´nchez-Eugenia and Sonia
Lo´pez (UBF) for their technical assistance in TrV purifi-
cation. Jon Agirre was supported by a UPV/EHU contract
(UPV-IT-461-07). Gerardo A. Marti is a researcher of the
CONICET, Argentina. Diego M. A. Gue´rin is researcher of
the Fundacio´n Biof´ısica Bizkaia and was partially supported
by Grants SAIOTEK, Gobierno Vasco (GV), and MICINN,
Spain. This work was partially supported by Accio´n Especial
AE-2009-1-21, from the GV, Spain, and CYTED 209RT0364
action (RedTrV: http://www.redtrv.org/), both granted to
Diego M. A. Gue´rin.
References
[1] B. C. Bonning and W. A. Miller, “Dicistroviruses,” Annual
Review of Entomology, vol. 55, pp. 129–150, 2010.
[2] O. A. Muscio, J. L. la Torre, and E. A. Scodeller, “Characteri-
zation of Triatoma virus, a picorna-like virus isolated from the
triatomine bug Triatoma infestans,” Journal of General Virology,
vol. 69, no. 11, pp. 2929–2934, 1988.
[3] O. A. Muscio, J. L. la Torre, M. A. Bonder, and E. A. Scodeller,
“Triatoma virus pathogenicity in laboratory colonies of Tri-
atoma infestans (Hemiptera: Reduviidae),” Journal of Medical
Entomology, vol. 34, no. 3, pp. 253–256, 1997.
[4] G. S. Rozas-Dennis and N. J. Cazzaniga, “Effects of Triatoma
virus (TrV) on the fecundity andmoulting of Triatoma infestans
(Hemiptera: Reduviidae),” Annals of Tropical Medicine and
Parasitology, vol. 94, no. 6, pp. 633–641, 2000.
[5] J. F. B. Querido, J. Agirre, G. A. Marti, D. M. A. Gue´rin, and
M. S. Silva, “Inoculation of Triatoma Virus (Dicistroviridae:
Cripavirus) elicits a non-infective immune response in mice,”
Parasites & Vectors, vol. 6, article 66, 2013.
[6] J. Agirre, K. Aloria, J. M. Arizmendi et al., “Capsid protein
identification and analysis of mature Triatoma virus (TrV)
virions and naturally occurring empty particles,” Virology, vol.
409, no. 1, pp. 91–101, 2011.
[7] G. A. Marti, E. Neumann, D. M. A. Rey et al., “AC-ELISA
and RT-PCR assays for diagnosis of Triatoma virus (TrV)
in triatomines (Hemiptera: Reduviidae) species,” Archives of
Virology, vol. 153, pp. 1427–1432, 2008.
[8] J. Agirre, G. Goret, M. LeGoff et al., “Cryo-transmission
electronmicroscopy reconstructions of triatoma virus particles:
a clue to unravel genome delivery and capsid disassembly,”
Journal of General Virology, 2013.
[9] M. Bostina, H. Levy, D. J. Filman, and J. M. Hogle, “Poliovirus
RNA is released from the capsid near a twofold symmetry axis,”
Journal of Virology, vol. 85, no. 2, pp. 776–783, 2011.
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Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

Servicio de Difusión de la Creación Intelectual

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013, Article ID 218593, 3 pageshttp://dx.doi.org/10.1155/2013/218593Methodology ReportMolecular Techniques for Dicistrovirus Detection withoutRNA Extraction or PurificationJailson F. B. Querido,1 Jon Agirre,2 Gerardo A. Marti,3Diego M. A. Guérin,2 and Marcelo Sousa Silva11 Centre for Malaria and Tropical Diseases, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,Rua da Junqueira, 100, 1349-008 Lisboa, Portugal2 Unidad de Biof´ısica (UBF), CSIC-UPV/EHU, Barrio Sarriena S/N, Bizkaia, 48940 Leioa, Spain3 Centro de Estudios Parasitolo´gicos y de Vectores (CEPAVE), CCT-La Plata/CONICET-UNLP, No. 584, 1900 La Plata, ArgentinaCorrespondence should be addressed to Marcelo Sousa Silva; mssilva@ihmt.unl.ptReceived 10 January 2013; Revised 15 March 2013; Accepted 21 March 2013Academic Editor: Zhirong SunCopyright © 2013 Jailson F. B. Querido et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insectpests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the mainvectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samplesof triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.1. IntroductionTriatoma virus (TrV), together with Solenopsis invicta virus-1, is one of the two members of the Dicistroviridae familypathogenic to insects of medical importance [1]. Because ofits horizontal way of transmission and high pathogenicityin populations of triatomines, TrV has been postulated tobe used as a biological control agent for both domiciliatedand nondomiciliated vectors of Chagas disease. It replicateswithin the cytoplasm of gut cells of triatomines [2], causing ahigh mortality rate, delayed development, and a reduction inthe fecundity [3, 4]. Recently, we have shown that this virusis unable to replicate nor is infective in mice and probably isnot infective to other vertebrates, reinforcing the possibilityof this virus to be used as a tool in biological control oftriatomines transmitters of Chagas disease [5].Dicistroviruses are characterized for having two openreading frames, ORF1 andORF2.TheORF2 is responsible forencoding the structural proteins VP1, VP2, VP3, and VP4,which in the case of TrV have respective masses of 29.7 kDa,28.4 kDa, 31.8 kDa, and 5.5 kDa [6]. The viral genome of TrVis a +ssRNA molecule of 9010 nucleotides [6].A reliable method for the detection of TrV by molec-ular techniques represents an important step towards theidentification of insect species being affected by this virusand the analysis of their geographical distribution as well.However, the diagnosis of triatomines in regions where it isdifficult to conduct scientific investigation poses a seriouschallenge. In this study, we present a new method that allowsthe reduction of costs and time required for the entire processof diagnosis, enabling the detection of TrV without resortingto RNA extraction, andwithout running the risk of degradingthe RNA during the extraction, purification and storageprocess.2. Materials and Methods2.1. Triatoma Virus Purification. The virus purification wasmade according to [6, 7]; dry feces coming from infectedinsects were collected on Whatman filter papers and excisedfrom them using a standard surgery scalpel until 2 g ofmaterial was collected. A buffer composed of 10mMNaCl,1mMMgCl2, and 200mM citric acid at pH 6 (Lysis buffer)2 BioMed Research Internationalwas added at a ratio of 100mL per gram of dry material.PMSF (N 98.5%, purchased from Sigma) thawed at −20∘Cin isopropanol (Merck, GR for analysis) was added up to afinal concentration of 1mM. The mixture was homogenizedfor 5min using a vortex mixer and then sonicated usinga Sanyo MSE Soniprep 150 operating at 10 s ON/10 s OFFfor 20 pulses. The homogenate was thereafter centrifuged ina Kontron T-1170 ultracentrifuge for 45min at 20,000 gavgusing a Beckman SW28.1 rotor. The collected supernatantwas then centrifuged for 2.5 h at 180,000 gavg using a Kon-tron TFT50.38 rotor. The resulting pellet was resuspendedovernight in 2mL of 10mMNaCl, 1mM MgCl2, and 50mMTris at pH 7.4 (NMTbuffer) and then loaded on top of a 35mLcontinuous 5%–30% sucrose gradient.The centrifugationwasat 100,000 gavg for 3 h in a Beckman SW28.1 rotor. Thegradient was fractionated from the bottom to the upper partin 0.5mL aliquots that were collected in Greiner 96-wellMasterBlocks and sealed thereafter with a silicone-based lid.The gradients were cooled in ice to avoid particle diffusionwithin them. The individual optical density of each aliquotwas then measured at both 260 nm and 280 nm using anImplen NanoPhotometer coupled with an LG100 UV-G6786cell. A baseline correction was performed using equivalentfractions from a control sucrose gradient. After plottingthe absorbance profile, selected aliquots were loaded onto12.5% polyacrylamide SDS-PAGE gels to check the purityof the samples. Following gradient fractionation, equivalentsamples were pooled together and dialyzed overnight against2 L of NMT buffer.2.2. Reverse Transcriptase-PCR. For the RT-PCR, we usedifferent concentration of purified TrV and feces samples(66.7mg/mL), without resorting to RNA extraction. Dryfeces coming from infected insects were resuspended in abuffer composed of 10mMNaCl, 1mMMgCl2, and 200mMcitric acid at pH 6 (Lysis buffer). PMSF (N 98.5%, purchasedfrom Sigma) thawed at −20∘C in isopropanol (Merck, GRfor analysis) was added up to a final concentration of 1mM.The homogenate was centrifuged in an Eppendorf cen-trifuge 5417 R for 30min at 20,000 gavg andwas subsequentlyfiltered through a 0.2 𝜇m. The feces sample was diluted inDEPC-treated water to a final concentration of 33.3mg/mL.As a positive control, we used viral RNA extracted (Tri-zol, Invitrogen) from purified TrV and feces samples. ThecDNA synthesis was performed according to cDNA Synthe-sis Kit Protocol (M-MuLV Reverse Transcriptase, ThermoScientific). In the PCR reactions were used single primerspairs: TrV sense: 5󸀠-TCAAAACTAACTATCATTCTGG-3󸀠(nt 7427 to 7448 in TrV ORF2 sequence) and TrV antisense:5󸀠-TTCAGCCTTATTCCCCCCC-3󸀠 (nt 8240–8258 in TrVORF2 sequence), with an expected product of 832 bp [8],selected from ORG2 region, conserved in dicistrovirus. PCRproducts were visualized on 1.5% agarose gels (runningfor 1 h at 100V) stained with ethidium bromide, and theirsizes were determined by comparison against DNA markers,HyperLadder I (Bioline, UK).M 1 2 3 4 5 6100002008001000(bp)Figure 1: PCR products on 1,5% agarose gel stained with ethidiumbromide. M: molecular weight marker 200 bp to 10000 bp (Bioline,UK). Lane 1: negative control, lane 2: 3000 ng of purified TrV, lane 3:300 ng of purified TrV, lane 4: 30 ng of purified TrV, lane 5: 3 ng ofpurified TrV, and lane 6: 0,3 ng of purified TrV.3. Results and DiscussionTo obtain the cDNA, we used full genome TrV particleswithout RNA extraction. We used a concentration rangebetween 3,000 and 0.3 ng of virus, being this concentrationmeasurement based on the viral proteins, diluted in DEPC-treated water. The cDNA amplifications were performedusing the selected primers pairs mentioned above. Weobtained amplification until a minimum concentration of3 ng of viral particles (Figure 1).To initiate the cDNA synthesis, we mixed 5 𝜇L of viralsamples (5, 3, and 1 𝜇L for feces samples) with 100 pmol ofOligo (dT), incubated first at 65∘C for 5min and then at4∘C for 5min. During the contact with the host cells, thevirus released the genome. In vitro, this process may occur byvarying certain environmental conditions such as pH or tem-perature [8, 9]. Incubating the virus at a temperature above65∘C will cause RNA release [8]. However, such temperatureand time of incubation should not be too long because 65∘C isclose to the melting temperature of RNA.The results indicatethat viral RNA can be detected in feces samples, after a singlecentrifugation and filtration step (Figure 2). These resultsreinforce the applicability and reproducibility of the methodpresented in the previous paragraph.Whenwe compare the result of RT-PCR from the samplesanalysed directly without resorting to RNA extraction tothe samples where RNA extraction was proceeded, we cansee that there is no significant difference between these twomethods, which indicates that the method proposed here is agood alternative to the method traditionally used.4. ConclusionsThe method proposed in this study allows the detectionof dicistrovirus by RT-PCR without resorting to RNABioMed Research International 3100001000800500100(bp)M 1 2 3 4 5 6 7 8 9 10 11Figure 2: PCR products on 1,5% agarose gel. M: molecular weightmarker 100 bp to 10000 bp (Jena Bioscience, Germany). Lane 1:negative control, lane 2: 5 𝜇L of viral RNA (from 3000 ng of purifiedTrV), lane 3: 5 𝜇L of viral RNA (from 300 ng of purified TrV), lane 4:5𝜇L of viral RNA (from 30 ng of purified TrV), lane 5: 5 𝜇L of viralRNA (from 0,03 ng of purified TrV), lane 6: 5 𝜇L of viral RNA (from1mg of feces), lane 7: 5 𝜇L of viral RNA (from 0.5mg of feces), lane8: 5𝜇L of viral RNA (from 0,25mg of feces), lane 9: 5𝜇L of fecessamples (without RNA extraction), lane 10: 3𝜇L of feces samples(without RNA extraction), and lane 11: 1 𝜇L of feces samples (withoutRNA extraction).extraction, which reduces the cost associated with thismethod and the risk of RNA degradation during the processof RNA extraction, and it is swimmingly reproducible in alow-budget laboratory. Moreover, this study provides newfacts about the thermostability of the TrV structure.AcknowledgmentsTheauthors are grateful to Rube´n Sa´nchez-Eugenia and SoniaLo´pez (UBF) for their technical assistance in TrV purifi-cation. Jon Agirre was supported by a UPV/EHU contract(UPV-IT-461-07). Gerardo A. Marti is a researcher of theCONICET, Argentina. Diego M. A. Gue´rin is researcher ofthe Fundacio´n Biof´ısica Bizkaia and was partially supportedby Grants SAIOTEK, Gobierno Vasco (GV), and MICINN,Spain. This work was partially supported by Accio´n EspecialAE-2009-1-21, from the GV, Spain, and CYTED 209RT0364action (RedTrV: http://www.redtrv.org/), both granted toDiego M. A. Gue´rin.References[1] B. C. Bonning and W. A. Miller, “Dicistroviruses,” AnnualReview of Entomology, vol. 55, pp. 129–150, 2010.[2] O. A. Muscio, J. L. la Torre, and E. A. Scodeller, “Characteri-zation of Triatoma virus, a picorna-like virus isolated from thetriatomine bug Triatoma infestans,” Journal of General Virology,vol. 69, no. 11, pp. 2929–2934, 1988.[3] O. A. Muscio, J. L. la Torre, M. A. Bonder, and E. A. Scodeller,“Triatoma virus pathogenicity in laboratory colonies of Tri-atoma infestans (Hemiptera: Reduviidae),” Journal of MedicalEntomology, vol. 34, no. 3, pp. 253–256, 1997.[4] G. S. Rozas-Dennis and N. J. Cazzaniga, “Effects of Triatomavirus (TrV) on the fecundity andmoulting of Triatoma infestans(Hemiptera: Reduviidae),” Annals of Tropical Medicine andParasitology, vol. 94, no. 6, pp. 633–641, 2000.[5] J. F. B. Querido, J. Agirre, G. A. Marti, D. M. A. Gue´rin, andM. S. Silva, “Inoculation of Triatoma Virus (Dicistroviridae:Cripavirus) elicits a non-infective immune response in mice,”Parasites & Vectors, vol. 6, article 66, 2013.[6] J. Agirre, K. Aloria, J. M. Arizmendi et al., “Capsid proteinidentification and analysis of mature Triatoma virus (TrV)virions and naturally occurring empty particles,” Virology, vol.409, no. 1, pp. 91–101, 2011.[7] G. A. Marti, E. Neumann, D. M. A. Rey et al., “AC-ELISAand RT-PCR assays for diagnosis of Triatoma virus (TrV)in triatomines (Hemiptera: Reduviidae) species,” Archives ofVirology, vol. 153, pp. 1427–1432, 2008.[8] J. Agirre, G. Goret, M. LeGoff et al., “Cryo-transmissionelectronmicroscopy reconstructions of triatoma virus particles:a clue to unravel genome delivery and capsid disassembly,”Journal of General Virology, 2013.[9] M. Bostina, H. Levy, D. J. Filman, and J. M. Hogle, “PoliovirusRNA is released from the capsid near a twofold symmetry axis,”Journal of Virology, vol. 85, no. 2, pp. 776–783, 2011.Submit your manuscripts athttp://www.hindawi.comHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Anatomy Research InternationalPeptidesInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporation http://www.hindawi.com International Journal ofVolume 2014ZoologyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Molecular Biology International GenomicsInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioinformaticsAdvances inMarine BiologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Signal TransductionJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioMed Research InternationalEvolutionary BiologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Biochemistry Research InternationalArchaeaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Genetics Research InternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Advances inVirolog yHindawi Publishing Corporationhttp://www.hindawi.comNucleic AcidsJournal ofVolume 2014Stem CellsInternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Enzyme ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014International Journal ofMicrobiology

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Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

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