High-throughput generation of transmissible antivirals

Effective population-level control of viral infections faces significant challenges including: how to therapeutically target the highest-risk populations, circumvent behavioral barriers, and overcome pathogen persistence and resistance mechanisms. A potential solution to overcome these barriers is the use of transmissible antivirals such as defective interfering particles (DIPs) or recently-proposed therapeutic interfering particles (TIPs). These transmissible antivirals are molecular parasites and transmit by ‘piggybacking’ on wildtype viral replication. By competing effectively for pools of common goods produced by the wildtype virus, DIPs/TIPs can interfere with wildtype virus replication and reduce viral loads in patients. As obligate parasites, TIPs would transmit via the same risk factors and transmission routes as wildtype viruses, automatically reaching high-risk populations, and thereby substantially limiting viral transmission even in resource-poor settings.At present, methods to generate such transmissible antivirals are ad hoc and rely on either expert knowledge to rationally design transmissible antivirals or a laborious and often lengthy process of prospecting for rare, spontaneously-occurring subgenomic mutants. Thus, while deletion mutants of human viruses are desired for use as viral vectors, live-attenuated vaccines, and transmissible antivirals, modern technologies to generate them at scale are not available.We introduce a new tool to overcome this barrier: a high-throughput method to generate diverse libraries of barcoded viral deletion mutants (> 1E5 unique mutants) at modest expense in a period of fewer than 5 days. The method is scalable and cyclical: viral strains with multiple deletions can be obtained by iterating the process. As proof of concept, we demonstrate the construction and screening of libraries of > 23, 000 barcoded deletion mutants of HIV-1 and > 90, 000 barcoded deletion mutants of Zika virus (ZIKV). Through repeated in vitro passage and deep sequencing of the pooled viral mutants, we are able to comprehensively map the cis-acting elements of HIV-1 and ZIKV at single base resolution. Moreover, we are able to track the prevalence of each barcoded deletion mutant through in vitro passage, and identify a subset of deletion mutants that are efficiently mobilized and amplified by the wildtype virus.For HIV-1, our results recapitulate empirical reports of cis-acting elements in the literature. We identify four cis-acting regions in the HIV-1 genome which could not be complemented in trans: (1) 5' LTR through the matrix domain of Gag, (2) cPPT/CTS, (3) RREthrough SA7, (4) PPT through 3' LTR. Thus the minimal proviral size of an HIV-1 vector with two intact LTRs is approximately 2.6 kbp.For ZIKV, we identify two cis-acting regions: (1) 5' UTR though C, (2) NS2A through the 3' UTR. We show that deletions which induce frameshift of the common open reading frame do not persist, in agreement with a basic mechanism of flaviviral replication. These results suggest a model where Pr, M, E, and NS1 can be provided in trans, but not C, NS2A/B, NS3, NS4A/B, and NS5.Finally, we use the information garnered in the HIV-1 screen to construct a library of transmissible antivirals. Use a modular cloning strategy, we assemble a combinatorial library of multiply-deleted mutants that are composed of a subset of adaptive HIV deletion mutants. We find that mutants with deletions of the accessory region vif –vpu, when combined with gag/pol deletions, interfere with wildtype virus replication and transmit efficiently in single round studies. These results suggest a model of interference where transcriptional asymmetry allows this subset of deletion mutants to compete effectively for a common pool of capsid proteins provided by the wildtype virus.Taken in whole, we show that we have developed a framework for generating transmissible antivirals from first principles. The method is of general use in virology, where the technology can be used to generate live-attenuated vaccines, viral vectors, and replicons, as well as to understand fundamental principles of viral replication and genetics in diverse viral systems. It is of particular interest in emerging viral infections, where therapies must be quickly generated and deployed

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

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

RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements.

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

RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements.

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

The structure and repertoire of small interfering RNAs in Leishmania (Viannia) braziliensis reveal diversification in the trypanosomatid RNAi pathway

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 (LbrAGO1). In contrast to T. brucei, Leishmania siRNAs are sensitive to 3Ј end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2Ј-Omethyltransferase, which modifies small RNA 3Ј ends. Consistent with this observation,~20% of siRNA 3Ј ends carry non-templated uridines. 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 doublestranded 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

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

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