Summary Among trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in-depth analysis of small interfering RNAs (siRNAs) associated with L. braziliensis Argonaute1 (AGO1). In contrast to T. brucei, Leishmania siRNAs are sensitive to 3’-end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2’-O-methyltransferase, which modifies small RNA 3’ends. Consistent with this observation, ~20% of siRNA 3’ ends carry non-templated uridine. Thus siRNA biogenesis, and most-likely their metabolism, is different in these organisms. Similarly to T. brucei, putative mobile elements and repeats constitute the major Leishmania siRNA-producing loci and AGO1 ablation leads to accumulation of long transcripts derived from putative mobile elements. However, contrary to T. brucei, no siRNAs were detected from other genomic regions with the potential to form double-stranded RNA, namely sites of convergent transcription and inverted repeats. Thus, our results indicate that organism-specific diversification has occurred in the RNAi pathway during evolution of the trypanosomatid lineage.

Acoustofluidic devices for manipulating microparticles in fluids are appealing for biological sample processing due to their gentle and high-speed capability of sorting cell-scale objects. Such devices are generally limited to moving particles toward locations at integer fractions of the fluid channel width (1/2, 1/4, 1/6, etc.). In this work, we introduce a unique approach to acoustophoretic device design that overcomes this constraint, allowing us to design the particle focusing location anywhere within the microchannel. This is achieved by fabricating a second fluid channel in parallel with the sample channel, separated from it by a thin silicon wall. The fluids in both channels participate to create the ultrasound resonance, while only one channel processes the sample, thus de-coupling the fluidic and acoustic boundaries. The wall placement and the relative widths of the adjacent channels define the particle focusing location. We investigate the operating characteristics of a range of these devices to determine the configurations that enable effective particle focusing and separation. The results show that a sufficiently thin wall negligibly affects focusing efficiency and location compared to a single channel without a wall, validating the success of this design approach without compromising separation performance. Using these principles to design and fabricate an optimized device configuration, we demonstrate high-efficiency focusing of microspheres, as well as separation of cell-free viruses from mammalian cells. These "transparent wall" acoustic devices are capable of over 90% extraction efficiency with 10 μm microspheres at 450 μL min(-1), and of separating cells (98% purity), from viral particles (70% purity) at 100 μL min(-1).

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Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min−1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. The approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

AbstractIt has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback, and then barcoded for tracking via sequencing (i.e., Random-Deletion Library sequencing, RanDeL-seq). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., LTR, Ψ, and RRE) and surprisingly indicated that HIV-1’s central DNA flap (i.e., central polypurine tract, cPPT to central termination sequence, CTS) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the UTR termini, encompassing a large fraction of the non-structural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections.ImportanceRecent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutant that conditionally replicate. Identifying and engineering DIPs requires that viral cis- and trans-acting elements be accurately mapped. Here we introduce a high-throughput method (Random Deletion Library sequencing, RanDeL-seq) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central poly-purine tract (cPPT) and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes, but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback and then barcoded for tracking via sequencing (i.e., random deletion library sequencing [RanDeL-seq]). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., long terminal repeat [LTR], Ψ, and Rev response element [RRE]) and, surprisingly, indicated that HIV-1’s central DNA flap (i.e., central polypurine tract [cPPT] to central termination sequence [CTS]) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300-bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the untranscribed (UTR) termini, encompassing a large fraction of the nonstructural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants, enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections. IMPORTANCE Recent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutants that conditionally replicate. Identifying and engineering DIPs require that viral cis- and trans-acting elements be accurately mapped. Here, we introduce a high-throughput method (random deletion library sequencing [RanDeL-seq]) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central polypurine tract (cPPT), and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

PCR, sequenced and analysed for drug resistance mutations and subtype information. Results HIV-2 RNA was detected in 7 of 10 ART-naïve and 2 of 6 ART-experienced patients. Detectable HIV-2 viral loads in these patients ranged from below the lower limit of quantification (<2.35 log IU/ml) to 5.45 log IU/ml. One ART-experienced patient had M184V, K65R and Y115F mutations in RT sequences from both plasma and PBMC. There were no drug resistance mutations identified from ART-naïve samples. Conclusion This is the first study in Ghana to show evidence of mutations in HIV-2 strains from patients receiving HIV-1 targeted antiretrovirals. The results prompt monitoring of drug resistance to improve clinical management of HIV-2 infected patients.

RNA interference (RNAi) is a powerful tool for genetic analysis. While many Leishmania species lost the RNAi pathway deep in evolution, species within the Leishmania subgenus Viannia retain RNAi pathway genes. Correspondingly, introduction of constructs expressing dsRNA in L. (V.) braziliensis resulted in substantial down regulation of all target mRNAs tested. We are optimizing and developing protocols that will facilitate the use of RNAi and applied them to the study of the Leishmania flagellum. We developed vectors allowing rapid generation of stem‐loop (StL) constructs using Gateway (Invitrogen®) technology, and are working on methods for inducible RNAi and maximization of RNAi activity. The Leishmania promastigote flagellum contains paraflagellar rod (PFR); RNAi of genes encoding two major proteins PFR1 or PFR2 yielded parasites lacking the PFR and exhibiting motility defects. We probed the role of the intraflagellar transport pathway (IFT) by targeting four genes implicated in anterograde or retrograde trafficking. We were unable to recover transfectants bearing the respective StL constructs. The same StL constructs were transfected into an Argonaute null mutant which lacks a functional RNAi pathway. All IFT StL constructs were now successfully introduced into the Argonaute mutant. These data suggest that the IFT pathway is essential for the viability of L. braziliensis promastigotes.