Striking gold with Dengue virus detection

Dengue viruses have become one of the most dangerous pathogens of our time. Here James Carter and Malcolm Fraser discuss a novel Dengue virus detection method based on gold nanoparticles and its potential impact on virology

Dengue viruses (DENV) are one of the most dangerous and important pathogens in the world¹. These mosquito-borne viruses cause periodic explosive epidemics in many tropical and sub-tropical countries, leading to approximately 100 million infections and 20,000 deaths each year ^2-5. As if these statistics aren’t daunting enough, as a result of air travel, global warming, insecticide, resistance, and poor healthcare approximately half the world’s population remains at risk for DENV infection⁶. Dengue fever (DF), Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS) are endemic to tropical and subtropical regions of the world, but the collapse of effective vector control programs, rapid dispersal of viruses due to ease of global travel, and migration of humans to non-tropical regions has resulted in DENV outbreaks in areas that were once non-endemic to the dengue viruses⁷. The epidemiology listed above remains the primary motivation behind the rigorous development of vaccines, prophylactic treatments, field release studies of transgenic mosquitoes, and especially detection methods are so imperative. We recently published research highlighting the development of a DENV detection method that couples the RNA cleaving capabilities of DNAzymes to the aggregation properties of gold nanoparticles⁸. It is anticipated that full development of this approach will enable the detection of DENV in infected patients and mosquito swarms.

DENV are maintained in a cycle that involves humans and the globally disseminated Aedes aegypti mosquito⁹. Infection with one of four antigenically distinct, but genetically related DENV serotypes can result in dengue fever (DF) and/or potentially fatal dengue hemorrhagic fever (DHF) ¹⁰. These Dengue virus related diseases are characterized by high fever, often with enlargement of the liver, and in severe cases circulatory and respiratory failure³. There may also be gastritis accompanied by one or more of the following: associated abdominal pain, nausea, vomiting, or diarrhea¹¹. Some cases develop much milder symptoms which are often misdiagnosed as influenza, chikungunya, or other viral infection when no rash is present [12]. The progression of DHF to DSS is a subject of debate, but the strongest experimental evidence leads to the hypothesis that an intense immune response promotes a cascade of events that culminates with plasma leakage^{13, 14}. Co-circulation of multiple serotypes can increase the risk for DHF or DSS ¹⁵.

An ideal virus detection method distinguishes between Dengue and other diseases with similar symptoms, is highly sensitive during the acute stage of infection, demonstrates utility in detecting DENV before symptoms manifest, gives rapid results to provide early warning, be inexpensive, easy to use, stable at temperatures greater than 30°C for use in a field environment, and have a long shelf life¹⁶. DENV detection methods should also be able to distinguish between primary and secondary infections, and must show utility in epidemiological surveillance and outbreak investigations by allowing independent detection of each serotype ^16.

Currently effective methods for mosquito-borne virus detection of infected persons or mosquitoes are limited to plaque assays, quantitation of viral production through PCR and RT-PCR-based methods (e.g. Lanciotti PCR ^17-19) or antigen detection (e.g. NS1 antigen tests ²⁰. Many traditional detection assays, especially for Dengue viruses, are prone to storage and contamination issues. Collectively these assays lack portability, require specialized equipment and training, can take hours to days to complete and in the case of diagnostic applications can be performed only after symptoms have manifested.

As a base technology, DNAzymes coupled to gold nanoparticles are potentially a superior method for assay development due to their extended stability/shelf-life at room temperature and functionality at high operating temperatures. These catalytic DNAs have proven effectiveness in cleaving virus genomes^21-25. Beyond this initial application, the DNAzyme components may be easily redesigned to cleave other viral genomes of interest, significantly advancing the fields of virus diagnostics and mosquito surveillance by providing an assay that is more rapid, easier to use, with greater portability and versatility, and more cost effective than current DENV detection methods. Once fully developed, the simplicity and versatility of our DENV detection approach will make it ideal for field applications.

Although catalytic oligonucleotide-driven detection methods are a relatively novel technology, catalytic oligonucleotides (e.g. DNAzymes), when coupled to AuNPs, have displayed considerable success in this endeavor. For example, DNAzyme-AuNP conjugates have demonstrated impressive sensitivity in detecting metal ions or RNA^26-30.

Recently we published an article describing our efforts to address the need for a definitive DENV detection method [8] by addressing the necessity for a rapid, portable, and simple method of virus detection that requires no specialized training, education, or equipment by coupling the RNA targeting ability of a DENV-specific DNAzyme (DDZ) with the aggregation properties of gold nanoparticles (AuNP). Initial development of our detection method, DDZ-AuNP, established a colorimetric DENV detection method, capable of detecting DENV directly from Aedes albopictus C6/36 cell culture fluids in a matter of minutes, without RNA isolation procedures while distinguishing each of the four DENV serotypes ⁸.

The colorimetric detection of DENV by DDZ-AuNP can be divided into three phases: targeting/cleavage, activation of AuNPs and aggregation/detection. In the presence of DENV, the 5’ and 3’ arms of the anti-DENV DNAzyme, DDZ, bind to the 3’ and 5’ ends of the targeted region. In the presence of the Mg²⁺ and heat DDZ digests the viral RNA ³¹, leading to aggregation of the DDZ bound AuNPs due to the presence of NaCl in the reaction mixture ^{32, 33}, allowing a rapid and visually or UV/Vis Spectrophotometry detectable red to clear/colorless color transition^{32, 33}. This color transition signifies the successful detection of DENV.

Further experimentation demonstrated we can successfully detect as little as 10¹ DENV TCID50 units/ml in cultured C6/36 cells.  Additional experimentation is in progress to fully elucidate the capabilities and limitations of our detection assay. Nevertheless, we are greatly encouraged by these results since we can detect DENV approximately 6.5 orders of magnitude below the viremia of patients who present with symptoms of DENV infection [34]. Moreover, our experimental results suggest the detection of DENV in mosquito populations is possible due to the low titer of infectious units we were able to detect.

The ability to detect DENV in the presence of so few infectious units may be principally due to the presence of immature/inactive virions^35-37, and RNA species^38-41 that are not detected by TCID₅₀-IFA, or even RT-PCR. For example, DENV and other viruses produce aberrant RNA species called “defective RNAs”^{38, 42}. These RNAs contain defects in the form of intra-genic stop codons, nucleotide insertions, or deletions, rendering many virions produced non-infectious ^{38, 41, 43}.  Some of these defective RNAs appear to be maintained during natural cycles of transmission, potentially due to complementation with fully functional DENV RNA genomes ^{38, 41}. Our Dengue virus colorimetric detection method, DDZ-AuNP, takes advantage of the presence of immature/inactive virions and aberrant RNA species due to the presence of detergent in the reaction mixture, the catalytic nature of DNAzymes and the affect of this RNA-induced catalysis on AuNP aggregation dynamics.

The utility of DDZ-AuNP as a DENV detection method is dependent on the availability of DENV RNA for targeting/digestion by DNAzymes and the ability to detect the catalysis.  These events are achieved through the presence of NaCl and SDS in the reaction mixture. NaCl, is an essential component of AuNP colorimetric detection assays because it drives aggregation of oligonucleotide-conjugated AuNPs, but only following the interaction of the AuNP conjugated oligonucleotides with the complimentary targets ^{44, 45}.  Regarding our DDZ-AuNP DENV detection method, NaCl driven aggregation appears to occur only in the presence of active DNAzymes. Although we speculate this is due to the conformational change of the DNAzyme during catalysis, the exact rationale is currently under investigation.

Liberating the DENV RNA genome from mature/immature/inactive virions increases the efficiency of our DDZ-AuNP detection assay. A cost effective, safe RNA extraction reagent that is stable in the reaction buffer and does not interfere with assay components would be ideal. Sodium dodecyl sulfate (SDS) fits this criteria since it is an effective non ionic detergent with proven effectiveness in lysing virus particles ⁴⁶. SDS may also be considered an ideal component for our colorimetric detection assays because it does not require additional manipulation during lysis.

A primary requirement for an optimal DENV detection assay is that it can effectively detect all four DENV serotypes ^16. We have to date demonstrated that our single DDZ-AuNP can detect all four DENV serotypes with a single detection method while discriminating between DENV serotypes. The versatility of DNAzymes allows us to design the binding arms, required for targeting and catalysis of the RNA substrate, for serotype-specific detection. For example, the DDZ-1 DNAzyme was designed to allow base pairing and subsequent catalysis of a region of the DENV genome that is fully conserved within the DENV-1 serotype only. Upon conjugation of DDZ-1 to AuNPs we were able to detect in a DENV-1 serotype specific manner.

Although in its infancy as a base detection technology, we are hopeful that the full development of our DENV detection method, DDZ-AuNP, will enable the identification of DENV in mosquito populations and patient samples.  Due to the versatility of DNAzymes and AuNPs, the development of detection systems using these entities can give rise to the development of novel techniques for detection of additional viral pathogens significantly advancing the fields of virus diagnostics and mosquito surveillance by providing an assay that is more rapid, easier to use, possess greater portability, and is more cost effective than current DENV detection methods.

Author: James R. Carter and Malcolm J. Fraser, Jr.

Contact: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana 46556, USA

 References

1. Clyde K, Kyle JL, Harris E: Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J Virol 2006, 80:11418-11431. 2. Randolph SE, Rogers DJ: The arrival, establishment and spread of exotic diseases: patterns and predictions. Nat Rev Microbiol 2010, 8:361-371. 3. Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, Vorndam AV: Dengue and dengue haemorrhagic fever. Lancet 1998, 352:971-977. 4. Pialoux G, Gauzere BA, Jaureguiberry S, Strobel M: Chikungunya, an epidemic arbovirosis. Lancet Infect Dis 2007, 7:319-327. 5. Weaver SC, Reisen WK: Present and future arboviral threats. Antiviral Res 2010, 85:328-345. 6.Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, et al: The global distribution and burden of dengue. Nature, 496:504-507. 7. Kalayanarooj S: Clinical Manifestations and Management of Dengue/DHF/DSS. Trop Med Health, 39:83-87. 8. Carter JR, Balaraman V, Kucharski CA, Fraser TS, Fraser MJ, Jr.: A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches. Virol J, 10:201. 9. Roberts L: Mosquitoes and disease. Science 2002, 298:82-83. 10. Qi RF, Zhang L, Chi CW: Biological characteristics of dengue virus and potential targets for drug design. Acta Biochim Biophys Sin (Shanghai) 2008, 40:91-101. 11. Kurane I, Takasaki T: Dengue fever and dengue haemorrhagic fever: challenges of controlling an enemy still at large. Rev Med Virol 2001, 11:301-311. 12. Tanner L, Schreiber M, Low JG, Ong A, Tolfvenstam T, Lai YL, Ng LC, Leo YS, Thi Puong L, Vasudevan SG, et al: Decision tree algorithms predict the diagnosis and outcome of dengue fever in the early phase of illness. PLoS Negl Trop Dis 2008, 2:e196. 13. Noisakran S, Perng GC: Alternate hypothesis on the pathogenesis of dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) in dengue virus infection. Exp Biol Med (Maywood) 2008, 233:401-408. 14. Chen JP, Cosgriff TM: Hemorrhagic fever virus-induced changes in hemostasis and vascular biology. Blood Coagul Fibrinolysis 2000, 11:461-483. 15. Halstead SB: Pathogenesis of dengue: challenges to molecular biology. Science 1988, 239:476-481. 16. Peeling RW, Artsob H, Pelegrino JL, Buchy P, Cardosa MJ, Devi S, Enria DA, Farrar J, Gubler DJ, Guzman MG, et al: Evaluation of diagnostic tests: dengue. Nat Rev Microbiol 2010, 8:S30-38. 17. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV: Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992, 30:545-551. 18. Gubler DJ: Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998, 11:480-496. 19. Guzman MG, Kouri G: Advances in dengue diagnosis. Clin Diagn Lab Immunol 1996, 3:621-627. 20. Dussart P, Labeau B, Lagathu G, Louis P, Nunes MR, Rodrigues SG, Storck-Herrmann C, Cesaire R, Morvan J, Flamand M, Baril L: Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum. Clin Vaccine Immunol 2006, 13:1185-1189. 21. Ryoo SR, Jang H, Kim KS, Lee B, Kim KB, Kim YK, Yeo WS, Lee Y, Kim DE, Min DH: Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown. Biomaterials, 33:2754-2761. 22. Appaiahgari MB, Vrati S: DNAzyme-mediated inhibition of Japanese encephalitis virus replication in mouse brain. Mol Ther 2007, 15:1593-1599. 23. Xie YY, Zhao XD, Jiang LP, Liu HL, Wang LJ, Fang P, Shen KL, Xie ZD, Wu YP, Yang XQ: Inhibition of respiratory syncytial virus in cultured cells by nucleocapsid gene targeted deoxyribozyme (DNAzyme). Antiviral Res 2006, 71:31-41. 24. Ren N, Wang F, Niu JQ: [A study of 10-23 DNAzyme inhibiting the expression of hepatitis B virus genes]. Zhonghua Gan Zang Bing Za Zhi 2005, 13:745-748. 25. Zhao CA, Zhao XD, Yu HG, Wu YP, Yang XQ: [Inhibition of respiratory syncytial virus replication in cultured cells by RNA-cleaving DNAzyme]. Zhonghua Er Ke Za Zhi 2003, 41:594-597. 26. Liu J, Cao Z, Lu Y: Functional nucleic acid sensors. Chem Rev 2009, 109:1948-1998. 27. Sun LQ, Cairns MJ, Gerlach WL, Witherington C, Wang L, King A: Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme. J Biol Chem 1999, 274:17236-17241. 28. Todd AV, Fuery CJ, Impey HL, Applegate TL, Haughton MA: DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format. Clin Chem 2000, 46:625-630. 29. Wu P, Hwang K, Lan T, Lu Y: A DNAzyme-gold nanoparticle probe for uranyl ion in living cells. J Am Chem Soc, 135:5254-5257. 30. Zhang Q, Cai Y, Li H, Kong DM, Shen HX: Sensitive dual DNAzymes-based sensors designed by grafting self-blocked G-quadruplex DNAzymes to the substrates of metal ion-triggered DNA/RNA-cleaving DNAzymes. Biosens Bioelectron, 38:331-336. 31. Ogawa A, Maeda M: Simple and rapid colorimetric detection of cofactors of aptazymes using noncrosslinking gold nanoparticle aggregation. Bioorg Med Chem Lett 2008, 18:6517-6520. 32. Song KM, Cho M, Jo H, Min K, Jeon SH, Kim T, Han MS, Ku JK, Ban C: Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer. Anal Biochem 2011. 33. Englebienne P: Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes. Analyst 1998, 123:1599-1603. 34. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis FA, Nisalak A: Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000, 181:2-9. 35. van der Schaar HM, Rust MJ, Chen C, van der Ende-Metselaar H, Wilschut J, Zhuang X, Smit JM: Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathog 2008, 4:e1000244. 36. van der Schaar HM, Rust MJ, Waarts BL, van der Ende-Metselaar H, Kuhn RJ, Wilschut J, Zhuang X, Smit JM: Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking. J Virol 2007, 81:12019-12028. 37. Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der Ende-Metselaar H, Lei HY, Wilschut J, Smit JM: Immature dengue virus: a veiled pathogen? PLoS Pathog, 6:e1000718. 38. Li D, Lott WB, Lowry K, Jones A, Thu HM, Aaskov J: Defective interfering viral particles in acute dengue infections. PLoS One, 6:e19447. 39. Juarez-Martinez AB, Vega-Almeida TO, Salas-Benito M, Garcia-Espitia M, De Nova-Ocampo M, Del Angel RM, Salas-Benito JS: Detection and sequencing of defective viral genomes in C6/36 cells persistently infected with dengue virus 2. Arch Virol, 158:583-599. 40. Ke R, Aaskov J, Holmes EC, Lloyd-Smith JO: Phylodynamic analysis of the emergence and epidemiological impact of transmissible defective dengue viruses. PLoS Pathog, 9:e1003193. 41. Aaskov J, Buzacott K, Thu HM, Lowry K, Holmes EC: Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes. Science 2006, 311:236-238. 42. Marriott AC, Dimmock NJ: Defective interfering viruses and their potential as antiviral agents. Rev Med Virol, 20:51-62. 43. Wang WK, Lin SR, Lee CM, King CC, Chang SC: Dengue type 3 virus in plasma is a population of closely related genomes: quasispecies. J Virol 2002, 76:4662-4665. 44. Ogawa A: RNA aptazyme-tethered large gold nanoparticles for on-the-spot sensing of the aptazyme ligand. Bioorg Med Chem Lett, 21:155-159. 45. Cao YC, Jin R, Mirkin CA: Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 2002, 297:1536-1540. 46. Becker R, Helenius A, Simons K: Solubilization of the Semliki Forest virus membrane with sodium dodecyl sulfate. Biochemistry 1975, 14:1835-1841.