One-step pentaplex real-time polymerase chain reaction assay for detection of zika, dengue, chikungunya, West nile viruses and a human housekeeping gene

•Accurate diagnosis is needed for clinically indistinguishable arboviral infections.•Establishment of differential and rapid diagnosis of ZIKV, DENV, CHIKV, and WNV.•Simultaneous detection of all ZIKV strains, all four DENV serotypes, CHIKV, WNV. Recent emergence of Zika virus (ZIKV), and the global spread of dengue (DENV), chikungunya (CHIKV) and West Nile viruses (WNV) raised urgent need of accurate and affordable molecular diagnosis of these clinically indistinguishable arboviral infections. We established a pentaplex real-time reverse transcription PCR (rRT-PCR) assay (CII-ArboViroPlex rRT-PCR) for specific and sensitive detection of the African and American genotypes of ZIKV, all four serotypes of DENV, CHIKV, WNV and a housekeeping gene as internal control in single reaction. Specific primers and probe sets were designed for ZIKV, DENV, CHIKV, WNV and RNase P (housekeeping gene) and tested for in-vitro transcribed RNA standards, virus cultures, clinical samples positive for ZIKV, DENV, CHIKV and WNV and limit of detection (LOD) were determined for each. Results Using ten-fold serially diluted in-vitro transcribed RNA, CII- ArboViroPlex rRT-PCR assay has LOD of 100 RNA copies/reaction (Rn) for ZIKV in serum or urine, 100 RNA copies/Rn for DENV in serum, and 10 RNA copies/Rn for CHIKV and WNV in serum. LODs from sera spiked with quantitated viral stocks were 2.6 × 102 GEQ/Rn for ZIKV, 2.2 × 101 GEQ/Rn for DENV-1, 9.4 × 100 GEQ/Rn for DENV-2, 2.3 × 102 GEQ/Rn for DENV-3, 1.4 × 103 GEQ/Rn for DENV-4, 2.7 × 102 GEQ/Rn for CHIKV, and 1.05 × 101 GEQ/Rn for WNV. The CII-ArboViroPlex rRT-PCR assay is a quantitative one-step pentaplex rRT-PCR assay for the molecular detection and differential diagnosis of ZIKV, DENV, CHIKV, WNV and a human housekeeping gene control in a single- PCR reaction

HLA-independent T cell receptors for targeting tumors with low antigen density.

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance