Performance Assessment of ATCC Quantitative Synthetic Analytical Reference Material for Zika Virus

Zika virus (ZIKV) is an enveloped, single-stranded RNA arbovirus in the Flaviviridae family. It is primarily transmitted via infected Aedes aegypti mosquitoes, sexual contact, or from mother to fetus during pregnancy. ZIKV is classified into two major genotypes: African and Asian. Most infections are asymptomatic or mild, with rash, fever, conjunctivitis, headache, malaise, and muscle or joint pain while infections during pregnancy are linked to severe congenital abnormalities like microcephaly. ZIKV infection has also been associated with neurological complications such as Guillain–Barré syndrome, though these outcomes are rare. The rapid geographic spread of ZIKV and the emergence of microcephaly and other neurological disorders led the World Health Organization (WHO) to declare ZIKV a Public Health Emergency of International Concern in 2016. This shift from enzootic virus to global health crisis highlights the impact of viral evolution, vector adaptation, and human ecological change on emerging infectious diseases.

Because ZIKV symptoms resemble other viral infections, accurate identification is critical. While culture-based approaches can be used for detection, viral growth and purification are tedious, slow, and costly. PCR-based methods provide sensitive, rapid alternatives but require high-quality reference materials. To meet this need, ATCC developed a quantitative synthetic analytical reference material for ZIKV (ATCC® VR-3252SD™). This proprietary construct incorporates key genomic regions targeted in published assays, spanning the membrane glycoprotein precursor (M), envelope (E), NS1, NS2B, NS3, NS4B, and NS5 segments.

ATCC® VR-3252SD™ was validated by next-generation sequencing, and RNA copy number (genome copies/µL) was quantified using digital-based PCR. We assessed its application using publicly available qPCR assays published by the CDC, WHO, and Faye et al. 2013 (Institut Pasteur of Dakar). Amplification of the synthetic RNA was compared with quantitative genomic RNA from Asian (ATCC® VR-1843DQ™) and African strains (ATCC® VR-1838DQ™). Ten-fold serial dilutions ranging from 50 to 50,000 genome copies/reaction were used to generate a standard curve of synthetic RNA. The synthetic RNA demonstrated an R² ≥ 0.999 and efficient amplification with an average slope of −3.443. Overall, these data demonstrate the applicability of ATCC® VR-3252SD™ as a reliable analytical reference material for the development and validation of molecular-based detection and quantification assays.