Assay optimization for molecular detection of Zika virus

Introduction The Zika virus is a mosquito-borne flavivirus with an approximately 11 kilobase ribonucleic acid (RNA) genome. (1) The virus usually causes a mild infection in adults, symptoms include fever, arthralgia and rash. (2,3) However, severe complications can occur, such as Guillain-Barre syndrome, (4) meningoencephalitis, (5) hearing loss and uveitis. (6,7) In the current Zika virus outbreak, intrauterine infections have been associated with fetal malformations. (8-11) Reliable detection of the Zika virus in infected people is key to understanding the epidemiology, the pathogenesis and alternative transmission routes of the virus, such as sexual intercourse and blood transfusions. (12) However, in areas where the Zika virus is co-circulating with dengue and chikungunya viruses, physicians cannot reliably diagnose the Zika virus infection by clinical presentation, because the viruses cause similar symptoms. Using serology for Zika virus diagnostics can be challenging because of the cross-reactivity of antibodies elicited by other endemic flaviviruses--such as dengue, yellow fever, St Louis encephalitis and West Nile viruses. (3,13,14) Molecular detection of viral nucleic acid using real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay is a highly reliable diagnostic method during acute infection. Currently, there are six widely used real-time RT-PCR assays for Zika virus detection. (7,13,15,16) The Pan American Health Organization (PAHO) has recommended an additional real-time RT-PCR assay. (12) High real-time RT-PCR sensitivity is important to avoid false-negative results. Nucleotide mutations in the binding sites of primers and probes can affect the sensitivity. (17) So far, the genetic variability of the Zika virus Asian lineage causing the current American outbreak is limited to about 2% nucleotide differences across the viral genome (Fig. 1). However, mutations do not occur evenly across viral genomes. Up to 10 nucleotide mismatches between the oligonucleotide sequences of the published assays and the Asian lineage consensus sequence already exist and in individual primers or probes, there are up to five mismatches (Fig. 2). Note that these are worst-case scenarios based on the genetic variability permitted within the Asian Zika virus lineage, with no single known Zika virus strain accumulating all of these mismatches. However, the increasing number of divergent Zika virus outbreak strains highlights the genetic variability as a potential limiting factor of the sensitivity of Zika virus real-time RT-PCR-based diagnostics. Here, we compare the sensitivity of published real-time RT-PCR assays and two new assays, which we designed to have less nucleotide mismatches with the current outbreak strains. We also present data on viral load profiles in blood and urine from infected patients, using one of the new assays. [FIGURE 1 OMITTED] Methods Assays We compared nine different assays. We included all Zika virus real-time RT-PCR assays published until 1 April 2016. These assays target the membrane (M), envelope (E), nonstructural protein (NS) 1, NS2b, NS3 and NS5 genomic domains. (7,12,13,15,16) We designed two new assays covering the currently known Zika virus genetic variability in the E and NS1 genomic domains (Table 1). These novel assays showed up to four potential mismatches per assay (Fig. 2) and were designed to avoid mismatches in the most critical 3'-terminal regions of oligonucleotides that affect primer binding the most. (17,19) The new NS1 assay was additionally designed to allow cross-detection of the Spondweni virus--the closest relative of the Zika virus--because regions conserved between related virus taxa are expected to have less variation than other genomic regions. Controls All controls are based on a current Zika virus outbreak strain (GenBank[R] accession number KU321639). As positive controls, we generated five assayspecific quantified in vitro transcripts (IVT) for the respective genomic target regions. …