Single-Cell Infection of Influenza A Virus Using Drop-Based Microfluidics.

Drop-based microfluidics has revolutionized single-cell studies and can be applied toward analyzing tens of thousands to millions of single cells and their products contained within picoliter-sized drops. Drop-based microfluidics can shed insight into single-cell virology, enabling higher-resolution analysis of cellular and viral heterogeneity during viral infection. In this work, individual A549, MDCK, and siat7e cells were infected with influenza A virus (IAV) and encapsulated into 100-µm-size drops. Initial studies of uninfected cells encapsulated in drops demonstrated high cell viability and drop stability. Cell viability of uninfected cells in the drops remained above 75%, and the average drop radii changed by less than 3% following cell encapsulation and incubation over 24 h. Infection parameters were analyzed over 24 h from individually infected cells in drops. The number of IAV viral genomes and infectious viruses released from A549 and MDCK cells in drops was not significantly different from bulk infection as measured by reverse transcriptase quantitative PCR (RT-qPCR) and plaque assay. The application of drop-based microfluidics in this work expands the capacity to propagate IAV viruses and perform high-throughput analyses of individually infected cells. IMPORTANCE Drop-based microfluidics is a cutting-edge tool in single-cell research. Here, we used drop-based microfluidics to encapsulate thousands of individual cells infected with influenza A virus within picoliter-sized drops. Drop stability, cell loading, and cell viability were quantified from three different cell lines that support influenza A virus propagation. Similar levels of viral progeny as determined by RT-qPCR and plaque assay were observed from encapsulated cells in drops compared to bulk culture. This approach enables the ability to propagate influenza A virus from encapsulated cells, allowing for future high-throughput analysis of single host cell interactions in isolated microenvironments over the course of the viral life cycle.

Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes.

The miniaturization of polymerase chain reaction (PCR) using drop-based microfluidics allows for amplification of single nucleic acids in aqueous picoliter-sized drops. Accurate data collection during PCR requires that drops remain stable to coalescence during thermocycling and drop contents are retained. Following systematic testing of known PCR additives, we identified an optimized formulation of 1% w/v Tween-20, 0.8 µg/µL bovine serum albumin, 1 M betaine in the aqueous phase, and 3 wt % (w/w) of the polyethylene glycol-perfluoropolyether2 surfactant in the oil phase of 50 µm diameter drops that maintains drop stability and prevents dye transport. This formulation enables a method we call off-chip drop reverse transcription quantitative PCR (OCD RT-qPCR) in which drops are thermocycled in a qPCR machine and sampled at various cycle numbers "off-chip", or outside of a microfluidic chip. qPCR amplification curves constructed from hundreds of individual drops using OCD RT-qPCR and imaged using epifluorescence microscopy correlate with amplification curves of ≈300,000 drops thermocycled using a qPCR machine. To demonstrate the utility of OCD RT-qPCR, influenza A virus (IAV) RNA was detected down to a single viral genome copy per drop, or 0.320 cpd. This work was extended to perform multiplexed detection of IAV M gene RNA and cellular ß-actin DNA in drops, and direct amplification of IAV genomes from infected cells without a separate RNA extraction step. The optimized additive formulation and the OCD-qPCR method allow for drop-based RT-qPCR without complex devices and demonstrate the ability to quantify individual or rare nucleic acid species within drops with minimal processing.

Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin.

A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.

Interleukin-7, but not thymic stromal lymphopoietin, plays a key role in the T cell response to influenza A virus.

The immune response to viral infection is ideally rapid and specific, resulting in viral clearance and establishment of immune memory. Some viruses such as HIV can evade such responses leading to chronic infection, while others like Influenza A can elicit a severe inflammatory response with immune-related complications including death. Cytokines play a major role in shaping the appropriate outcomes to infection. While Interleukin-7 (IL-7) has a critical role in T and B cell development, treatment with IL-7 has recently been shown to aid the adaptive T cell response in clearance of chronic viral infection. In contrast, the IL-7-related cytokine thymic stromal lymphopoietin (TSLP) has a limited role in lymphocyte development but is important in the immune response to parasitic worms and allergens. The role for these cytokines in the immune response to an acute viral infection is unclear. IL-7 and TSLP share IL-7Rα as part of their heterodimeric receptors with the gamma common chain (γc) and TSLPR, respectively. We investigated the role of IL-7 and TSLP in the primary immune response to influenza A infection using hypomorphic IL-7Rα (IL-7Rα(449F)) and TSLPR(-/-) mice. We found that IL-7, but not TSLP, plays an important role in control of influenza A virus. We also showed that IL-7 signaling was necessary for the generation of a robust influenza A-specific CD4 and CD8 T cell response and that this requirement is intrinsic to CD8 T cells. These findings demonstrate a significant role for IL-7 during acute viral infection.

Temporal- and strain-specific host microRNA molecular signatures associated with swine-origin H1N1 and avian-origin H7N7 influenza A virus infection.

MicroRNAs (miRNAs) repress the expression levels of genes by binding to mRNA transcripts, acting as master regulators of cellular processes. Differential expression of miRNAs has been linked to virus-associated diseases involving members of the Hepacivirus, Herpesvirus, and Retrovirus families. In contrast, limited biological and molecular information has been reported on the potential role of cellular miRNAs in the life cycle of influenza A viruses (infA). In this study, we hypothesize that elucidating the miRNA expression signatures induced by low-pathogenicity swine-origin infA (S-OIV) pandemic H1N1 (2009) and highly pathogenic avian-origin infA (A-OIV) H7N7 (2003) infections could reveal temporal and strain-specific miRNA fingerprints during the viral life cycle, shedding important insights into the potential role of cellular miRNAs in host-infA interactions. Using a microfluidic microarray platform, we profiled cellular miRNA expression in human A549 cells infected with S- and A-OIVs at multiple time points during the viral life cycle, including global gene expression profiling during S-OIV infection. Using target prediction and pathway enrichment analyses, we identified the key cellular pathways associated with the differentially expressed miRNAs and predicted mRNA targets during infA infection, including the immune system, cell proliferation, apoptosis, cell cycle, and DNA replication and repair. By identifying the specific and dynamic molecular phenotypic changes (microRNAome) triggered by S- and A-OIV infection in human cells, we provide experimental evidence demonstrating a series of temporal and strain-specific host molecular responses involving different combinatorial contributions of multiple cellular miRNAs. Our results also identify novel potential exosomal miRNA biomarkers associated with pandemic S-OIV and deadly A-OIV-host infection.

Polar positioning of a conjugation protein from the integrative and conjugative element ICEBs1 of Bacillus subtilis.

ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.