Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus.

BACKGROUND: The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources. METHODS: We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively. RESULTS: These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/µl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83). CONCLUSIONS: The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas.

The increased prevalence of neuropathogenic strains of EHV-1 in equine abortions.

A panel of 426 archived EHV-1 isolates collected (1951-2006) from equine abortions was analyzed using a real-time Taq-Man((R)) allelic discrimination PCR assay. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A(2254)) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G(2254)) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A(2258)-->C(2258)) in ORF30, in addition to A(2254)-->G(2254). Interestingly, the non-neuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A(2254)-->G(2254) and A(2258)-->C(2258) substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.