Deficiency of the circadian clock transcriptional factor BMAL1 results in the development of premature aging in mice. In agreement with the accelerated aging phenotype, we observed an increase in t...

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain–Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Epidemics caused by the reemergence of Zika virus (ZIKV) warrant the need to develop new diagnostic measures to complement currently used detection methods. In this study, we explored the detection of ZIKV antigen in a defined leukocyte subset from patients’ whole-blood specimens. Whole-blood samples were obtained at the acute and early convalescent phases from ZIKV-infected patients during the Singapore outbreak in August–September 2016. Presence of ZIKV antigen was determined by flow cytometry staining for intracellular ZIKV NS3, using a ZIKV-specific polyclonal antibody. The presence of ZIKV antigen was determined in CD45⁺CD14⁺ monocytes. Data showed that ZIKV NS3 antigen could be detected in CD45⁺CD14⁺ monocytes. The levels of detection were further categorized into 3 groups: high (positivity among >40% of monocytes), moderate (positivity among 10%–40%), and low (positivity among <10%). While a majority of patients showed a decrease in the amount of ZIKV antigen detected at later time points, some patients displayed higher levels as the disease progressed. Our data highlights an alternative approach in using flow cytometry as a sensitive method for detecting ZIKV antigen in whole blood. Importantly, it further confirms the role of CD14⁺ monocytes as an important cellular target for ZIKV infection during the viremic phase.