Attomolar Zika virus oligonucleotide detection based on
loop-mediated isothermal ampli
fication and AC susceptometry
Bo Tian
a, Zhen Qiu
a, Jing Ma
b, Teresa Zardán Gómez de la Torre
a, Christer Johansson
c,
Peter Svedlindh
a, Mattias Strömberg
a,na
Department of Engineering Sciences, Uppsala University, The Ångström Laboratory, Box 534, SE-751 21 Uppsala, Sweden
b
Department of Immunology, Genetics and Pathology, Uppsala University, The Rudbeck Laboratory, SE-751 85 Uppsala, Sweden
c
Acreo Swedish ICT AB, Arvid Hedvalls backe 4, SE-411 33 Göteborg, Sweden
a r t i c l e i n f o
Article history: Received 28 April 2016 Received in revised form 16 June 2016
Accepted 28 June 2016 Available online 29 June 2016 Keywords:
Zika virus
Loop-mediated isothermal amplification Magnetic nanoparticles
Brownian relaxation AC susceptometer
a b s t r a c t
Because of the serological cross-reactivity among theflaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are drama-tically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27 min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.
& 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Zika virus (ZIKV), the causative agent of the infectious disease Zika fever, is a positive-sense RNA virus that belongs to the family Flaviviridae, genus Flavivirus, which is related to dengue fever, yellow fever, Japanese encephalitis and West Nile fever (Kuno et al., 1998;Sikka et al., 2016). Until recently, Zika fever was con-sidered mild and self-limited and therefore has received less at-tention (Haug et al., 2016;Petersen et al., 2016). However, it has recently been found that ZIKV infection could trigger Guillain-Barré syndrome (Cao-Lormeau et al., 2016;Broutet et al., 2016); and could also be associated with microcephaly fetal death (Fauci and Morens, 2016;Brasil et al., 2016). ZIKV is mainly transmitted by day-time active female Aedes aegypti and Aedes albopictus mosquitoes (Sikka et al., 2016), but can also be transmitted sexu-ally (Foy et al., 2011;Venturi et al., 2016;Oster et al., 2016;D ’Or-tenzio et al., 2016), from mother to the fetus (Calvet et al., 2016;
Melo et al., 2016; Schuler-Faccini et al., 2016), and potentially through blood transfusion (Musso et al., 2014;Marano et al., 2016). The recent outbreaks of ZIKV have drawn the world's attention
towards this relatively unstudied virus, particularly for its asso-ciation with the increased number of newborns presenting mi-crocephaly reported in Brazil (Campos et al., 2015; Rodriguez-Morales, 2015;Heukelbach et al., 2016).
The incubation period of Zika fever ranges from three to twelve days after the bite of an infected mosquito, which is similar to other infections caused byflaviviruses (Goeijenbier et al., 2016;
Sikka et al., 2016), and the clinical signs persist for two to seven days (Korzeniewski et al., 2016). Laboratory diagnostics of ZIKV are based mainly on the analysis of serum, by using viral RNA and/or antibody based detection methods (Haug et al., 2016;Al-Qahtani et al., 2016). Due to serological cross-reactivity amongflaviviruses in IgM-antibody based assays, diagnostics based on molecular testing are considered more reliable (Haug et al., 2016). The cur-rent prevalent molecular detection method is reverse-transcrip-tion polymerase chain reacreverse-transcrip-tion (RT-PCR), which can detect 320 copies/
μ
L (approximately 530 aM) ZIKV RNA in a total assay time (excluding the time for RNA extraction) of about 90 min (Faye et al., 2008;Faye et al., 2013;Musso et al., 2015). However, since viremia decreases over time, RT-PCR based methods should be performed during thefirst seven days after symptom onset (Haug et al., 2016;Buathong et al., 2015), when the ZIKV loads are re-ported at the level of approximately 1 fM in bodyfluid samples (Lanciotti et al., 2008;Gourinat et al., 2015; Barzon et al., 2016); Contents lists available atScienceDirectjournal homepage:www.elsevier.com/locate/bios
Biosensors and Bioelectronics
http://dx.doi.org/10.1016/j.bios.2016.06.085
0956-5663/& 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
nCorresponding author.
after that, reliable results can only be provided by labor-intensive serologic testing (Z four days after symptom onset, and IgM an-tibodies persist from two weeks to several months) (Petersen et al., 2016). Therefore, more sensitive and reliable molecular de-tection methods are required to meet the challenge of ZIKV daily diagnoses on blood donators, pregnant women and travelers from areas with ongoing ZIKV transmission.
Among the reported isothermal molecular detection techni-ques that have the potential to replace traditional PCR/RT-PCR, loop-mediated isothermal amplification (LAMP) has become an excellent option due to its high amplification efficiency, specificity, low cost, time-saving protocol and the ability of amplifying non-denatured DNA samples (Notomi et al., 2000;Tomita et al., 2008). In our previous work (Tian et al., 2016b), we reported a LAMP based optomagnetic biosensor, which could detect 10 aM synthetic Newcastle disease virus DNA in 30 min. It has been demonstrated that Bst 3.0 polymerase could amplify both RNA and DNA se-quences under identical conditions, and the LAMP-optomagnetic biosensor was verified by performing tests on clinical RNA samples (vaccine and tissue specimens) (Tian et al., 2016b). The LAMP-optomagnetic assay was performed in a three-step procedure in-cluding amplification, labeling and read-out. However, due to the optical influence of the Mg2P2O7 precipitation, the LAMP-opto-magnetic detection procedure required opening of the reaction tube after the LAMP reaction, which greatly increased the risk of carryover contamination (Tomita et al., 2008).
Here, we present a simple and homogeneous ZIKV detection method that combines LAMP and a magnetic AC susceptibility readout method based on the response of the magnetic nano-particle (MNP) system, and the three step procedure is reduced to a two-step procedure. Streptavidin-coated 100 nm magnetic na-noparticles were mixed with LAMP reagents including biotinylated inner primers prior to the reaction. During the LAMP reaction, the hydrodynamic volume of the MNPs dramatically increased due to binding between the streptavidin groups on the particles and the biotinylated amplicons. Mg2P2O7, a by-product produced in a successful LAMP reaction (Mori et al., 2001), was also involved in the increase of the hydrodynamic volume of the MNPs. The in-crease in hydrodynamic volume of the MNPs resulted in a Brow-nian relaxation frequency shift to lower frequency which was subsequently measured by a portable AC susceptometer (Astalan et al., 2004). An increased target oligonucleotide concentration resulted in a larger frequency shift to lower frequency. The LAMP-AC susceptometer biosensor achieves a limit of detection (LOD) of 1 aM synthetic ZIKV oligonucleotide with a total assay time of 27 min. The same sensitivity was achieved when performing the test in 20% serum samples. From these results we can hopefully widen the detection window of ZIKV viremia and contribute to the Zika fever control.
2. Materials and methods 2.1. Reagents
Bst 3.0 polymerase, isothermal amplification buffer II and MgSO4were purchased from New England Biolabs (Ipswich, UK). dNTP mix was purchased from Thermo Fisher Scientific (Waltham, USA). Fetal bovine serum, D-biotin and MQ water were purchased from Sigma-Aldrich. Streptavidin modified 100 nm MNPs (multi-core magnetic beads containing clusters of small single domain particles, 10 mg/mL, 6 1012 particles/mL) were purchased from Micromod (Rostock, Germany). For the serum sample detection, 10 nM synthetic ZIKV oligonucleotide was serial diluted with 20% fetal bovine serum.
2.2. Primer design
RNA-dependent-RNA-polymerase (NS5) sequences of 51 ZIKV strains from the outbreak in Easter Island in 2014 (GenBank ac-cession number: KM078929-KM078979) (Tognarelli et al., 2016) were compared and a 230 bp highly conserved region (Table S1) was chosen as the target gene for primer design. LAMP primers (inner primer pair FIP/BIP, outer primer pair F3/B3 and loop primer pair LF/LB) were designed using Primer Explorer version 4 (https:// primerexplorer.jp/e/), and the sequences are shown in Table S1. The inner primer FIP was labeled with a biotin group at the 5′-end. The target sequence and six primers were synthesized by In-tegrated DNA Technologies (Coralville, USA).
2.3. LAMP reaction
A 100
μ
L reaction mixture contained 200μ
g/mL MNPs (strep-tavidin-coated MNPs or biotin-blocked MNPs), 1.6μ
M inner pri-mers, 0.2μ
M outer primers, 0.4μ
M loop primers, 1 isothermal amplification buffer II (containing 2 mM MgSO4), 6 mM MgSO4 (8 mM in total), 32 U Bst 3.0 polymerase, 1.4 mM dNTPs and 40μ
L of analyte solution (synthetic ZIKV oligonucleotide). The mixture was incubated at 69°C (metal bath) for 12 min2.4. AC susceptometry measurement
After incubation, the reaction tube was mounted in a portable commercial AC susceptometer, DynoMags(Acreo Swedish ICT AB, Göteborg, Sweden), to measure the frequency-dependent AC sus-ceptibility. Sixteen data points were measured in the frequency range of 4–100 Hz, and the readout time was 15 min. The fre-quency-dependent out-of-phase component of the susceptibility, χ″, was recorded. The position of the χ″peak in frequency is de-fined as the Brownian relaxation frequency fB, which is given by
πη
= ( )
fB k TB / 6 Vh, where k TB is the thermal energy,ηis the dynamic
viscosity andVhis the hydrodynamic volume of the relaxing entity.
2.5. Optimization of LAMP reaction
Agarose gel electrophoresis analysis was employed to evaluate the influence of different reaction temperatures (61, 63, 65, 67, 69 and 71°C) and different inner primer concentrations (0.4, 0.8 and 1.6
μ
M). Synthetic ZIKV oligonucleotide at a concentration of 1 pM was used as analyte, and the Mg2P2O7 precipitations were re-moved by centrifugation after 12 min amplification. To find an optimum biotinylated inner primer to streptavidin-MNP ratio that could balance the reaction efficiency and the blocking effect caused by the excess of biotinylated primers, the inner primer concentration vs. streptavidin-MNP concentration was evaluated in the AC susceptometer by amplifying 1 fM synthetic ZIKV oligonucleotide.2.6. Scanning electron microscopy measurement
After the LAMP reaction, the MNPs (streptavidin-coated MNPs or biotin-blocked MNPs) suspended in the reaction mixture were separated by a magnetic stand, washed twice by 50
μ
L MQ water and resuspended in 25μ
L MQ water. A 100μ
L control reaction mixture (positive LAMP reaction without MNPs) was centrifuged, washed twice with 50μ
L MQ water and resuspended in 25μ
L MQ water to collect the Mg2P2O7precipitation. The morphology and nanostructure of the three samples (volume-amplified streptavi-din-coated MNP aggregates, volume-amplified biotin-blocked MNP aggregates and Mg2P2O7precipitation) were characterized by scanning electron microscopy (SEM, Zeiss-1530) using an in-lensdetector for secondary electrons and the microscope was operated at 3 kV electron beam accelerating voltage.
3. Results and discussion
3.1. AC susceptometry detection and setup
An illustration of the LAMP based susceptometry biosassay is presented inFig. 1. Synthetic ZIKV oligonucleotides are mixed with streptavidin-MNPs and other LAMP reagents including biotiny-lated inner primers. Due to the binding of amplicons (and/or Mg2P2O7) during the LAMP reaction, the hydrodynamic volume of MNPs increases, which results in a shift in fBto lower frequency. 3.2. Optimization of LAMP reaction
Agarose gel electrophoresis analysis showed that the optimum reaction temperature was 69°C (Fig. S1A). At this temperature the LAMP reaction produced a maximum amount of amplicons and therefore this temperature was employed in the following tests. Although Fig. S1Bshows that the reaction efficiency can be im-proved by increasing the concentration of inner primers, it may be anticipated that the higher amount of biotinylated inner primer would lead to a smaller fB shift, since the polymerase would preferentially amplify the free inner primers instead of primers that are already bound to MNPs (according to the supplier, 200
μ
g/mL streptavidin-MNPs can be saturated with approxi-mately 0.02μ
M biotin groups). Therefore, the shift in fB was evaluated with respect to the inner primer concentration vs. streptavidin-MNP concentration. As shown inFig. S2, detection of 1 fM synthetic ZIKV oligonucleotides gave the largest fBshift with 1.6μ
M inner primers and 200μ
g/mL MNPs.Considering that the biotin-streptavidin reaction is much faster than the LAMP reaction, the streptavidin groups on the surface of MNPs can be saturated by biotinylated primers before the primers being amplified. Moreover, polymerases that bind to the surface of MNPs contribute more to the MNP volume amplification. There-fore, a higher MNP concentration results in a lower amount of polymerases on each MNP, and consequently in less amplicons on each MNP (Fig. S2). The amplification time was also optimized and this was done by turbidity-based naked-eye detection (data not shown). Due to the high amplification efficiency and the end-point detection format, signals of positive samples reach the saturation level easily and are then not useful for quantification. Therefore, a longer reaction time results in a narrower dynamic detection range. An amplification time of 12 min was chosen to achieve
rapid attomolar target detection without narrowing the dynamic detection range.
3.3. Quantitative synthetic ZIKV oligonucleotide detection
Serial dilutions of synthetic ZIKV oligonucleotides, ranging from 1 pM to 100 zM, were amplified in presence of streptavidin-MNPs and analyzed by the AC susceptometer; the spectra ofχ″are shown inFig. 2A, showing that fBshifts to lower values with in-creasing analyte concentration. Three independent measurements were performed for each analyte concentration. Since we focus more on the peak position, the χ″spectra were not normalized with respect to χ′∞(high-frequency value of the in-phase com-ponent of the volume susceptibility) as done in previous work (Zardán Gómez de la Torre et al., 2011). The typical spectra of the three independent measurements are shown in this work, and fB are extracted from each measurement for the following analysis.
Interestingly, the lowest fB observed in this work is 7.6 Hz (Fig. 2A, 1 pM target sequence), which is much lower than ob-served in previous work (approximately 36 Hz, 1 pM target se-quence) (Tian et al., 2016b). The frequency peak obtained by the optomagnetic setup is not exactly equal but very close to the fB (Donolato et al., 2015;Tian et al., 2016a). The difference between the protocol used in this work and the one used in previous work is that in the previous work the biotinylated amplicons were in-cubated with MNPs after the LAMP reaction. Therefore, we hy-pothesize that the much larger shift in fB when using this new protocol is explained not only by binding of MNPs to amplicons, but also by the Mg2P2O7 precipitation that is formed during the LAMP reaction and deposited onto the surface of the MNPs.
To verify this hypothesis, we first investigated whether the biotin-blocked streptavidin-MNPs could be volume-amplified by incubating with already formed biotinylated amplicons or Mg2P2O7 precipitation. The biotinylated amplicons and the Mg2P2O7 pre-cipitation were separated by centrifugation after the LAMP reac-tion and incubated for 15 min at room temperature with biotin-blocked MNPs, respectively.Fig. S3shows that neither the bioti-nylated amplicons nor the precipitated Mg2P2O7 could obviously change fB of the biotin-blocked MNPs. This means that (1) the streptavidin groups on the MNPs were completely blocked; (2) the biotin-blocked MNPs have no interaction with the already formed LAMP products and byproducts; and (3) the viscosity is not ob-viously changed by the LAMP reaction.
Next we investigated whether biotin-blocked MNPs could be volume-amplified during the LAMP reaction. The biotin-blocked MNPs were mixed with LAMP reagents and the analyte, and re-acted with serial dilutions of synthetic ZIKV samples; the AC
Fig. 1. Illustration of the LAMP based susceptometry assay. Two independent steps, incubation and measurement, are included in the illustration from left to right. In-cubation; target oligonucleotides are amplified and the product/byproduct interacts with MNPs during the LAMP reaction. Measurement; the reaction tube is mounted in the AC susceptometer and measured (for clarity, the excitation coil is shown with a section in the middle removed to reveal the pickup coil).
susceptibility results are shown in Fig. 2B. A frequency shift is observed for ZIKV concentrations ≥ 100 aM, and the lowest ob-served fB is about 20 Hz (1 pM analyte). This result clearly de-monstrates that MNPs have an interaction with the Mg2P2O7 precipitation during its formation. This interaction was further confirmed by scanning electron microscopy (SEM).
The dose-response curves of fBvs. ZIKV oligonucleotide con-centration are shown inFig. 3 for triplicate measurements. The cut-off value was calculated as the average signal of the blank control samples minus three standard deviations, and the LOD was defined as the lowest ZIKV oligonucleotide concentration whose value is below the cut-off. The LOD and dynamic detection range of streptavidin-MNP based LAMP-AC susceptometry analysis were 1 aM and 1–103 aM, respectively. For the biotin-blocked MNP based analysis, MNPs were volume-amplified only by the inter-action with Mg2P2O7, and the sensitivity was reduced: an LOD of
32 aM was achieved. The average coefficients of variation in the dynamic detection range of streptavidin-MNP based and biotin-blocked MNP based analysis were 9.75% and 8.95%, respectively. 3.4. Scanning electron microscopy study
By using SEM, we further investigated the hypothesis that MNP can be volume-amplified by the deposition of Mg2P2O7during the LAMP. The MNPs were separated, washed and collected after the LAMP reaction for SEM analysis (1 pM target analyte). Due to the rigorous separation and washing procedure, the aggregates at-tracted by the magnetic stand are considered to be MNPs with strongly attached amplicons (for streptavidin-MNPs) and/or Mg2P2O7 precipitation (for both streptavidin-MNPs and biotin-blocked MNPs).Fig. 4shows the morphology and nanostructures of the collected volume-amplified MNP aggregates, as well as the precipitated Mg2P2O7that were collected from the control LAMP reaction without MNPs. Compared to the regular nanostructures of the Mg2P2O7precipitation (Fig. 4C), magnetic aggregates based on volume-amplified streptavidin-MNPs (Fig. 4A) and volume-am-plified biotin-blocked MNP (Fig. 4B) contain a dense mass of ir-regular Mg2P2O7nanostructures. The existence of these irregular Mg2P2O7nanostructures or Mg2P2O7fragments implies that MNPs are involved in the formation of Mg2P2O7nanostructures and have a strong interaction with them. It is thus confirmed that the Mg2P2O7precipitation occurs on the MNP surface during the LAMP reaction, and results in an additional shift of fB. This phenomenon also implies that other magnetic nanostructures can possibly serve as labels of LAMP products without surface functionalization, which can be further utilized to establish Mg2P2O7 deposition based biosensors.
3.5. Spiked serum sample detection
Since blood analysis is a routine work in ZIKV diagnosis and control, serum samples were tested by the proposed streptavidin-MNP based LAMP-AC susceptometry assay. The matrix effects of serum can slow down the amplification and reduce the sensitivity of the biosensor. After evaluation of several serum concentrations, it was found that serum concentrations lower than 20% have no obvious impact on the sensitivity of proposed system. Therefore, synthetic ZIKV oligonucleotide samples were spiked into 20% serum to simulate five times diluted pure serum samples, and
Fig. 2. Typical χ″ spectra for the indicated concentrations of synthetic ZIKV oligonucleotide. (A) Streptavidin-MNP based LAMP-AC susceptometry analysis. (B) Biotin-blocked MNP based LAMP-AC susceptometry analysis.
Fig. 3. Dose-response curves for synthetic ZIKV oligonucleotide detected by streptavidin-MNP based (black circles) and biotin-blocked MNP based (red squares) LAMP-AC susceptometry analysis. Error bars indicate one standard deviation based on three independent measurements. Cut-off values are indicated by the horizontal lines. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)
analyzed by the streptavidin-MNP based LAMP-AC susceptometry system. The spectra and dose-response curve of 20% serum de-tection are shown inFig. 5. While the LOD (1 aM synthetic ZIKV oligonucleotide) is preserved for the 20% serum samples, the dy-namic detection range (approximately four orders of magnitude, from 1 aM to 104 aM) is larger than for samples without serum (approximately three orders of magnitude,Fig. 3). We attribute the extended dynamic detection range to the matrix effects of the serum, which can slow down the reaction but not influence the equilibrium (the lowest fB is 7.9 Hz, nearly the same as for the sample without serum). The average coefficient of variation in the dynamic detection range was 7.73% for 20% serum detection. The result shows that detection on 20% serum samples can achieve an LOD of 1 aM with a total assay time of 27 min (12 min LAMP and 15 min readout), which is two orders of magnitude more sensitive and faster than the reported RT-PCR based ZIKV detection.
3.6. Specificity evaluation
The specificity (selectivity) of the proposed streptavidin-MNP based LAMP-AC susceptometry assay was investigated by con-sidering four types of synthetic NS5 gene from relevant viruses (Dengue virus, GenBank accession number: EF595819; Yellow fe-ver virus, GenBank accession number: AY541441; Japanese en-cephalitis virus, GenBank accession number: FJ515937; and West Nile virus, GenBank accession number: AY187015) as negative controls (synthesized by Integrated DNA Technologies). The syn-thetic oligonucleotides were spiked into 20% serum at a con-centration of 1 pM. Results presented inFig. S4 show that the responses from the negative controls are similar to the blank control, indicating a high specificity of the proposed method. 4. Conclusion
This work presents afirst report of a LAMP-AC susceptometry assay for rapid and highly sensitive quantitative ZIKV detection.
Fig. 4. Scanning electron microscopy images of volume-amplified streptavidin-MNP aggregates (A), volume-amplified biotin-blocked MNP aggregates (B) and precipitated Mg2P2O7nanostructures (C).
Fig. 5. 20% serum sample measurements. (A) Typical χ″ spectra for the indicated concentrations of synthetic ZIKV oligonucleotide. (B) Dose-response curve of streptavidin-MNP based LAMP-AC susceptometry analysis. Error bars indicate one standard deviation based on three independent measurements. The cut-off value is indicated by the horizontal line.
The proposed detection system recognizes 1 aM synthetic ZIKV oligonucleotide in 27 min. The assay has a dynamic detection range of 1 aM-10 fM targets in 20% serum sample. Although we only detected synthetic Zika virus DNA, we have demonstrated in a previous paper that the enzyme (Bst 3.0 polymerase) could am-plify both RNA and DNA sequences under identical conditions, and performs well when detecting clinical RNA samples (vaccine and tissue specimens) (Tian et al., 2016b). This method is approxi-mately two orders of magnitude more sensitive and faster than the reported RT-PCR based ZIKV serum detection (Faye et al., 2008;
Faye et al., 2013), and is thus a promising method for ZIKV diag-nosis and control. In future work, a heating unit will be integrated into the AC susceptometer in order to automate and simplify the detection procedure.
Acknowledgments
This work was financially supported by Swedish Research Council Formas (Project nos. 221–2012-444, 221–2014-574 and 2011–1692).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version athttp://dx.doi.org/10.1016/j.bios.2016.06.085.
References
Al-Qahtani, A.A., Nazir, N., Al-Anazi, M.R., Rubino, S., Al-Ahdal, M.N., 2016. J. Infect. Dev. Ctries. 10 (3), 201–207.
Astalan, A.P., Ahrentorp, F., Johansson, C., Larsson, K., Krozer, A., 2004. Biosens. Bioelectron. 19 (8), 945–951.
Barzon, L., Pacenti, M., Berto, A., Sinigaglia, A., Franchin, E., Lavezzo, E., Brugnaro, P., Palu, G., 2016. Eur. Surveill. 21 (10), 2–6.
Brasil, P., Pereira Jr., J.P., Raja Gabaglia, C., Damasceno, L., Wakimoto, M., Ribeiro Nogueira, R.M., Carvalho de Sequeira, P., Machado Siqueira, A., Abreu de Car-valho, L.M., Cotrim da Cunha, D., Calvet, G.A., Neves, E.S., Moreira, M.E., Ro-drigues Baiao, A.E., Nassar de Carvalho, P.R., Janzen, C., Valderramos, S.G., Cherry, J.D., Bispo de Filippis, A.M., Nielsen-Saines, K., 2016. N. Engl. J. Med. Broutet, N., Krauer, F., Riesen, M., Khalakdina, A., Almiron, M., Aldighieri, S., Espinal,
M., Low, N., Dye, C., 2016. N. Engl. J. Med. 374 (16), 1506–1509.
Buathong, R., Hermann, L., Thaisomboonsuk, B., Rutvisuttinunt, W., Klungthong, C., Chinnawirotpisan, P., Manasatienkij, W., Nisalak, A., Fernandez, S., Yoon, I.K., Akrasewi, P., Plipat, T., 2015. Am. J. Trop. Med. Hyg. 93 (2), 380–383.
Calvet, G., Aguiar, R.S., Melo, A.S., Sampaio, S.A., de Filippis, I., Fabri, A., Araujo, E.S., de Sequeira, P.C., de Mendonca, M.C., de Oliveira, L., Tschoeke, D.A., Schrago, C. G., Thompson, F.L., Brasil, P., Dos Santos, F.B., Nogueira, R.M., Tanuri, A., de Fi-lippis, A.M., 2016. Lancet Infect. Dis.
Campos, G.S., Bandeira, A.C., Sardi, S.I., 2015. Emerg. Infect. Dis. 21 (10), 1885–1886.
Cao-Lormeau, V.M., Blake, A., Mons, S., Lastere, S., Roche, C., Vanhomwegen, J., Dub, T., Baudouin, L., Teissier, A., Larre, P., Vial, A.L., Decam, C., Choumet, V., Halstead, S.K., Willison, H.J., Musset, L., Manuguerra, J.C., Despres, P., Fournier, E., Mallet,
H.P., Musso, D., Fontanet, A., Neil, J., Ghawche, F., 2016. Lancet 387 (10027), 1531–1539.
D’Ortenzio, E., Matheron, S., de Lamballerie, X., Hubert, B., Piorkowski, G., Maquart, M., Descamps, D., Damond, F., Yazdanpanah, Y., Leparc-Goffart, I., 2016. N. Engl. J. Med.
Donolato, M., Antunes, P., Bejhed, R.S., Zardán Gómez de la Torre, T., Østerberg, F.W., Strömberg, M., Nilsson, M., Strømme, M., Svedlindh, P., Hansen, M.F., Vavassori, P., 2015. Anal. Chem. 87 (3), 1622–1629.
Fauci, A.S., Morens, D.M., 2016. N. Engl. J. Med. 374 (7), 601–604.
Faye, O., Faye, O., Diallo, D., Diallo, M., Weidmann, M., Sall, A.A., 2013. Virol. J. 10, 311.
Faye, O., Faye, O., Dupressoir, A., Weidmann, M., Ndiaye, M., Alpha Sall, A., 2008. J. Clin. Virol. 43 (1), 96–101.
Foy, B.D., Kobylinski, K.C., Chilson Foy, J.L., Blitvich, B.J., Travassos da Rosa, A., Haddow, A.D., Lanciotti, R.S., Tesh, R.B., 2011. Emerg. Infect. Dis. 17 (5), 880–882.
Goeijenbier, M., Slobbe, L., van der Eijk, A., de Mendonca Melo, M., Koopmans, M.P., Reusken, C.B., 2016. Neth. J. Med. 74 (3), 104–109.
Gourinat, A.C., O’Connor, O., Calvez, E., Goarant, C., Dupont-Rouzeyrol, M., 2015. Emerg. Infect. Dis. 21 (1), 84–86.
Haug, C.J., Kieny, M.P., Murgue, B., 2016. N. Engl. J. Med.
Heukelbach, J., Alencar, C.H., Kelvin, A.A., De Oliveira, W.K., Pamplona de Goes Cavalcanti, L., 2016. J. Infect. Dev. Ctries. 10 (2), 116–120.
Korzeniewski, K., Juszczak, D., Zwolinska, E., 2016. Int. Marit. Health 67 (1), 31–37.
Kuno, G., Chang, G.J., Tsuchiya, K.R., Karabatsos, N., Cropp, C.B., 1998. J. Virol. 72 (1), 73–83.
Lanciotti, R.S., Kosoy, O.L., Laven, J.J., Velez, J.O., Lambert, A.J., Johnson, A.J., Stanfield, S.M., Duffy, M.R., 2008. Emerg. Infect. Dis. 14 (8), 1232–1239.
Marano, G., Pupella, S., Vaglio, S., Liumbruno, G.M., Grazzini, G., 2016. Blood Transfus. 14 (2), 95–100.
Melo, A.S.O., Malinger, G., Ximenes, R., Szejnfeld, P.O., Sampaio, S.A., de Filippis, A. M.B., 2016. Ultrasound Obstet. Gynecol. 47 (1), 6–7.
Mori, Y., Nagamine, K., Tomita, N., Notomi, T., 2001. Biochem. Biophys. Res. Com-mun. 289 (1), 150–154.
Musso, D., Nhan, T., Robin, E., Roche, C., Bierlaire, D., Zisou, K., Yan, A.S., Cao-Lor-meau, V.M., Broult, J., 2014. Eur. Surveill. 19 (14), 6–8.
Musso, D., Roche, C., Nhan, T.X., Robin, E., Teissier, A., Cao-Lormeau, V.M., 2015. J. Clin. Virol. 68, 53–55.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Nucleic Acids Res. 28 (12), E63.
Oster, A.M., Brooks, J.T., Stryker, J.E., Kachur, R.E., Mead, P., Pesik, N.T., Petersen, L.R., 2016. Morb. Mortal. Wkly. Rep. 65 (5), 120–121.
Petersen, L.R., Jamieson, D.J., Powers, A.M., Honein, M.A., 2016. N. Engl. J. Med. 374 (16), 1552–1563.
Rodriguez-Morales, A.J., 2015. J. Infect. Dev. Ctries. 9 (6), 684–685.
Schuler-Faccini, L., Ribeiro, E.M., Feitosa, I.M.L., Horovitz, D.D.G., Cavalcanti, D.P., Pessoa, A., Doriqui, M.J.R., Neri, J.I., Neto, J.M.D., Wanderley, H.Y.C., Cernach, M., El-Husny, A.S., Pone, M.V.S., Serao, C.L.C., Sanseverino, M.T.V., Embry, B.M.G.S.Z., 2016. Morb. Mortal. Wkly. Rep. 65 (3), 59–62.
Sikka, V., Chattu, V.K., Popli, R.K., Galwankar, S.C., Kelkar, D., Sawicki, S.G., Stawicki, S.P., Papadimos, T.J., 2016. J. Glob. Infect. Dis. 8 (1), 3–15.
Tian, B., Bejhed, R.S., Svedlindh, P., Strömberg, M., 2016a. Biosens. Bioelectron. 77, 32–39.
Tian, B., Ma, J., Zardán Gómez de la Torre, T., Bálint, Á., Donolato, M., Hansen, M.F., Svedlindh, P., Strömberg, M., 2016b. (submitted for publication).
Tognarelli, J., Ulloa, S., Villagra, E., Lagos, J., Aguayo, C., Fasce, R., Parra, B., Mora, J., Becerra, N., Lagos, N., Vera, L., Olivares, B., Vilches, M., Fernandez, J., 2016. Arch. Virol. 161 (3), 665–668.
Tomita, N., Mori, Y., Kanda, H., Notomi, T., 2008. Nat. Protoc. 3 (5), 877–882.
Venturi, G., Zammarchi, L., Fortuna, C., Remoli, M.E., Benedetti, E., Fiorentini, C., Trotta, M., Rizzo, C., Mantella, A., Rezza, G., Bartoloni, A., 2016. Eur. Surveill. 21 (8), 2–5.
Zardán Gómez de la Torre, T., Mezger, A., Herthnek, D., Johansson, C., Svedlindh, P., Nilsson, M., Strømme, M., 2011. Biosens. Bioelectron. 29 (1), 195–199.
