Synthetic RNA for the Development and Evaluation of In Vitro Diagnostic Devices Designed to Detect Dengue, Chikungunya, and Zika

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Hematophagous arthropod vectors are responsible for the transmission of some of the most devastating diseases throughout the world. The World Health Organization estimates that vector-borne diseases account for over 17% of all infectious diseases, contributing to more than 1 billion cases and 1 million deaths annually.1 Accurate diagnosis of these diseases can be complicated due to a variety of factors, including analogous clinical presentation, serological cross-reactivity, or the possibility of co-infection. Thus, the accurate and rapid diagnosis of vector-borne diseases through validated diagnostic methods is critical in enabling prompt treatment and controlling microbial dissemination. Here, we discuss the co-circulation and potential co-infection of the mosquito-borne Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV) viruses; the challenges associated with the identification and discernment of these viruses; and the need for authenticated control materials for the validation of in vitro diagnostic tools.

Outbreaks of mosquito-borne diseases are common in tropical and subtropical climates in regions throughout the Americas, Africa, Asia, and the Pacific Islands. Environmental changes, unplanned urbanization, and the globalization of travel and trade have enhanced the spread of DENV, CHIKV, and ZIKV to many of these geographic areas, resulting in localized epidemics and potentiating viral co-circulation. Within these environments, transmission of DENV, CHIKV, and ZIKV between humans is facilitated via the Aedes aegypti and Aedes albopictus mosquito vectors.2-5 This overlap between affected areas combined with a shared competent vector has enabled the possibility of viral co-infection within a single host.6-9 However, given the similar clinical presentation (Table 1) of these viral strains, co-infections may not be readily identified and distinguishing between infectious agents based on symptoms alone may be complicated.

Table 1. Possible Symptoms of Dengue, Chikungunya, and Zika Infection3-5,10-12

Main Symptoms Dengue Chikungunya Zika


Skin rash

Muscle and joint pain

Nausea and/or vomiting

Bleeding or bruising

Pain behind the eyes


DENV, CHIKV, and ZIKV can be diagnosed through viral isolation, serological tests, molecular-based methods, or a combination of these techniques.13-15 Viral isolation enables the identification of an active infection and is considered to be a sensitive and specific form of detection. However, this method is laborious and can only be performed by personnel with technical expertise within laboratories equipped with the appropriate biosafety level infrastructure. In contrast, serological-based assays that detect the presence of anti-viral IgM antibodies are considerably more rapid and are often the go-to method of diagnosis. However, these assays are less sensitive and specific as it may take several days before IgM antibodies reach detectable levels within a patient; in addition, antibodies may still be at appreciable levels months following viral clearance, and serological cross-reactivity with similar viruses can occur.11,13,16-18 For example, in the detection of DENV, serological assays are not reliable until the fifth day following the onset of symptoms and antibodies can remain detectable 2-3 months following viral clearance.13,19 Further, cross-reactivity with other flaviviruses such as West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, yellow fever virus, and ZIKV have been reported.13,16

The molecular-based detection of viral RNA in serum via reverse transcription polymerase chain reaction (RT-PCR) is the preferred method for the early detection and confirmation of DENV, CHIKV, and ZIKV in clinical samples.20 This method enables rapid, reliable detection and quantification of viral load and is considerably more sensitive than serological methods and easier to perform than viral isolation. Within the last decade, there have been a number of molecular-based assays developed for the detection of DENV, CHIKV, and ZIKV in serum and/or mosquito samples, including multiplex RT-PCR assays for the simultaneous detection of DENV and CHIKV.21-30 More recently, single-reaction, multiplex RT-PCR assays have been developed with the ability to detect and distinguish all three viruses from the same sample, enabling the identification of a singular infection or co-infection within mosquitoes or clinical samples.31,32

To aid in the development and evaluation of molecular-based methods for the detection of DENV, CHIKV, and ZIKA, IVD manufacturers may rely on RNA preparations as positive control materials. However, as these viruses are challenging to cultivate and have specific biosafety level requirements, acquiring these materials can prove challenging. To circumvent these issues, ATCC has developed quantitative synthetic molecular standards under ISO 13485:2003 for CHIKV, DENV serotypes I-IV, and ZIKV comprising short conserved fragments that represent clinically relevant regions of the genome. As compared to genomic RNA preparations, synthetic molecular standards are easier to use as controls for RT-PCR assays, demonstrate a longer shelf-life, exhibit less variability, and eliminate the need for viral propagation. Further, they are compatible with various existing molecular assays, can be handled in a biosafety level one facility, are stable, and can be shipped internationally without the need for a specialized permit. Taken together, these synthetic molecular standards provide convenient, safe, and well characterized reference materials for use as control materials in the manufacture and validation of molecular-based assays.

Overall, vector-borne diseases represent a serious concern throughout the world. Rapid and reliable diagnostic assays are imperative in enabling prompt treatment and managing viral transmission. However, similar clinical presentation, the possibility of co-infection, and serological cross-reactivity can complicate a diagnosis. To help support the need for validated IVDs designed for the detection of DENV, CHIKV, and ZIKV infection or co-infection, ATCC has developed synthetic molecular standards representing clinically relevant regions of the viral genome. These products provide convenient, safe materials that can be used as positive controls in assay development and validation.


  1. World Health Organization. Vector-borne Diseases Fact Sheet, 2016.
  2. Centers for Disease Control and Prevention. Surveillance and Control of Aedes aegypti and Aedes albopictus in the United States, 2016.
  3. World Health Organization. Chikungunya Fact Sheet N°327, 2015. 
  4. World Health Organization. Dengue and Severe Dengue Fact Sheet N°117, 2015. 
  5. World Health Organization. Zika Virus Fact Sheet, 2016. 
  6. Caron M. et al. Recent introduction and rapid dissemination of Chikungunya virus and Dengue virus serotype 2 associated with human and mosquito coinfections in Gabon, central Africa. Clin Infect Dis 55: e45-53, 2012. 
  7. Chang SF, et al. Concurrent isolation of chikungunya virus and dengue virus from a patient with coinfection resulting from a trip to Singapore. J Clin Microbiol 48:4586-4589, 2010. 
  8. Dupont-Rouzeyrol M, et al. Co-infection with Zika and dengue viruses in 2 patients, New Caledonia, 2014. Emerging infectious diseases 21: 381-382, 2015. 
  9. Villamil-Gomez WE, et al. Dengue, chikungunya and Zika co-infection in a patient from Colombia. J Infect Public Health 9(5): 684-686, 2016. 
  10. Centers for Disease Control and Prevention. Symptoms and What To Do If You Think You Have Dengue, 2012. 
  11. Centers for Disease Control and Prevention. Chikungunya Virus - Symptoms, Diagnosis, & Treatment, 2015. 
  12. Centers for Disease Control and Prevention. Zika Virus - Symptoms, Diagnosis, & Treatment, 2016. 
  13. Centers for Disease Control and Prevention. Dengue - Laboratory Guidance and Diagnostic Testing, 2016. 
  14. Centers for Disease Control and Prevention. Chikungunya virus - Diagnostic Testing, 2016. 
  15. World Health Organization & Pan American Health Organization. Zika virus (ZIKV) Surveillance in the Americas: Interim guidance for laboratory detection and diagnosis, 29 June 2015. 
  16. World Health Organization & Pan American Health Organization. Epidemiological Alert - Zika virus infection, 7 May 2015. 
  17. Parida M, et al. Rapid and Real-time Assays for Detection and Quantification of Chikungunya Virus. Future Virology 3: 179-192, 2008. 
  18. Hoz JM, et al. Fatal cases of Chikungunya virus infection in Colombia: Diagnostic and treatment challenges. J Clin Virol 69: 27-29, 2015. 
  19. Bhat VG, et al. Challenges in the Laboratory Diagnosis and Management of Dengue Infections. Open Microbiol J 9: 33-37, 2015. 
  20. Centers for Disease Control and Prevention. Revised diagnostic testing for Zika, chikungunya, and dengue viruses in US Public Health Laboratories, 2016. 
  21. Pongsiri P, et al. Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus. Asian Pac J Trop Med 5(5): 342-346, 2012. 
  22. Chen H, et al. Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses. J Mol Diagn 17(6): 722-728, 2015. 
  23. Faye O, et al. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes. Virol J 10: 311, 2013. 
  24. Faye O, et al. One-step RT-PCR for detection of Zika virus. J Clin Virol 43: 96-101, 2008. 
  25. Lanciotti RS, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 14: 1232-1239, 2008. 
  26. Pfeffer M, et al. Specific detection of chikungunya virus using a RT-PCR/nested PCR combination. J Vet Med 49: 49-54, 2002. 
  27. Panning M, et al. Performance of the RealStar Chikungunya virus real-time reverse transcription-PCR kit. J Clin Microbiol 47: 3014-3016, 2009. 
  28. Centers for Disease Control and Prevention. DENV-1-4 Real-Time RT-PCR Assay for Detection and Serotype Identification of Dengue virus. Instructions for Use Package Insert
  29. Conceicao TM, Da Poian AT, Sorgine MH. A real-time PCR procedure for detection of dengue virus serotypes 1, 2, and 3, and their quantitation in clinical and laboratory samples. J Virol Methods 163(1): 1-9, 2010. 
  30. Waggoner JJ, et al. Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses. PLoS Negl Trop Dis 7(4): e2116, 2013. 
  31. Johnson M. GenArraytion Launches Test for Mosquito-Borne Pathogens Amid New CDC Zika Testing Guidelines, February 5, 2016. 
  32. Johnson M. CDC Recommends Zika Testing for Some Pregnant Women; Labs Developing New MDx Assays, January 27, 2016.
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