Influenza A viral burst size from thousands of infected single cells using droplet quantitative PCR (dqPCR).

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses.

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.

Measuring colloid-surface interaction forces in parallel using fluorescence centrifuge force microscopy.

Interactions between colloidal-scale structures govern the physical properties of soft and biological materials, and knowledge of the forces associated with these interactions is critical for understanding and controlling these materials. A common approach to quantify colloidal interactions is to measure the interaction forces between colloids and a fixed surface. The centrifuge force microscope (CFM), a miniaturized microscope inside a centrifuge, is capable of performing hundreds of force measurements in parallel over a wide force range (10-2 to 104 pN), but CFM instruments are not widely used to measure colloid-surface interaction forces. In addition, current CFM instruments rely on brightfield illumination and are not capable of fluorescence microscopy. Here we present a fluorescence CFM (F-CFM) that combines both fluorescence and brightfield microscopy and demonstrate its use for measuring microscale colloidal-surface interaction forces. The F-CFM operates at speeds up to 5000 RPM, 2.5× faster than those previously reported, yielding a 6.25× greater maximum force than previous instruments. A battery-powered GoPro video camera enables real-time viewing of the microscopy video on a mobile device, and frequency analysis of the audio signal correlates centrifuge rotational speed with the video signal. To demonstrate the capability of the F-CFM, we measure the force required to detach hundreds of electrostatically stabilized colloidal microspheres attached to a charged glass surface as a function of ionic strength and compare the resulting force distributions with an approximated DLVO theory. The F-CFM will enable microscale force measurements to be correlated with fluorescence imaging in soft and biological systems.

Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation.

Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation. Here, we demonstrate that the P. aeruginosa gene PA4463 [hibernation promoting factor (HPF)], but not the ribosome modulation factor (PA3049), is required for ribosomal RNA preservation during prolonged nutrient starvation conditions. Single-cell-level studies using fluorescence in situ hybridization (FISH) and growth in microfluidic drops demonstrate that, in the absence of hpf, the rRNA abundances of starved cells decrease to levels that cause them to lose their ability to resuscitate from starvation, leaving intact nondividing cells. P. aeruginosa defective in the stringent response also had reduced ability to resuscitate from dormancy. However, FISH analysis of the starved stringent response mutant showed a bimodal response where the individual cells contained either abundant or low ribosome content, compared with the wild-type strain. The results indicate that ribosome maintenance is key for maintaining the ability of P. aeruginosa to resuscitate from starvation-induced dormancy and that HPF is the major factor associated with P. aeruginosa ribosome preservation.

Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics.

UNLABELLED: Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity. IMPORTANCE: RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.

Monodisperse Emulsion Drop Microenvironments for Bacterial Biofilm Growth.

In this work, microfluidic technology is used to rapidly create hundreds of thousands of monodisperse double and triple emulsion drops that serve as 3D microenvironments for the containment and growth of bacterial biofilms. The size of these drops, with diameters from tens to hundreds of micrometers, makes them amenable to rapid manipulation and analysis. This is demonstrated by using microscopy to visualize cellular differentiation of Bacillus subtilis biofilm communities within each drop and the bacterial biofilm microstructure. Biofilm growth is explored upon specific interfaces in double and triple emulsions and upon negative and positive radii of curvature. Biofilm attachment of matrix and flagella mutants is studied as well as biofilms of Pseudomonas aeruginosa. This is the first demonstration of biofilms grown in microscale emulsion drops, which serve as both templates and containers for biofilm growth and attachment. These microenvironments have the potential to transform existing high-throughput screening methods for bacterial biofilms.

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics.

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.

Nanoscale double emulsions stabilized by single-component block copolypeptides.

Water-in-oil-in-water emulsions are examples of double emulsions, in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase. Emulsions occur in many forms of processing and are used extensively by the foods, cosmetics and coatings industries. Because of their compartmentalized internal structure, double emulsions can provide advantages over simple oil-in-water emulsions for encapsulation, such as the ability to carry both polar and non-polar cargos, and improved control over release of therapeutic molecules. The preparation of double emulsions typically requires mixtures of surfactants for stability; the formation of double nanoemulsions, where both inner and outer droplets are under 100 nm, has not yet been achieved. Here we show that water-in-oil-in-water double emulsions can be prepared in a simple process and stabilized over many months using single-component, synthetic amphiphilic diblock copolypeptide surfactants. These surfactants even stabilize droplets subjected to extreme flow, leading to direct, mass production of robust double nanoemulsions that are amenable to nanostructured encapsulation applications in foods, cosmetics and drug delivery.

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.

Single-cell herpes simplex virus type 1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics.

Single-cell analyses of viral infections reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of herpes simplex virus type 1 (HSV-1) infection of neurons at the single-cell level. We used micrometer-scale Matrigel beads, termed microgels, to culture individual murine superior cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are encapsulated in individual media-in-oil droplets with a dual-fluorescent reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. These techniques for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

Starve a cold or feed a fever? Identifying cellular metabolic changes following infection and exposure to SARS-CoV-2.

Viral infections induce major shifts in cellular metabolism elicited by active viral replication and antiviral responses. For the virus, harnessing cellular metabolism and evading changes that limit replication are essential for productive viral replication. In contrast, the cellular response to infection disrupts metabolic pathways to prevent viral replication and promote an antiviral state in the host cell and neighboring bystander cells. This competition between the virus and cell results in measurable shifts in cellular metabolism that differ depending on the virus, cell type, and extracellular environment. The resulting metabolic shifts can be observed and analyzed using global metabolic profiling techniques to identify pathways that are critical for either viral replication or cellular defense. SARS-CoV-2 is a respiratory virus that can exhibit broad tissue tropism and diverse, yet inconsistent, symptomatology. While the factors that determine the presentation and severity of SARS-CoV-2 infection remain unclear, metabolic syndromes are associated with more severe manifestations of SARS-CoV-2 disease. Despite these observations a critical knowledge gap remains between cellular metabolic responses and SARS-CoV-2 infection. Using a well-established untargeted metabolomics analysis workflow, we compared SARS-CoV-2 infection of human lung carcinoma cells. We identified significant changes in metabolic pathways that correlate with either productive or non-productive viral infection. This information is critical for characterizing the factors that contribute to SARS-CoV-2 replication that could be targeted for therapeutic interventions to limit viral disease.

Single-cell Herpes Simplex Virus type-1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics.

Single-cell analyses of viral infections often reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of HSV-1 infection of neurons at the single-cell level. We used micron-scale Matrigel beads, termed microgels, to culture individual murine Superior Cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are subsequently enclosed in individual media-in-oil droplets with a dual fluorescent-reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. The innovative techniques presented here for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

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.

Granular Matrigel: restructuring a trusted extracellular matrix material for improved permeability.

Matrigel is a polymeric extracellular matrix material produced by mouse cancer cells. Over the past four decades, Matrigel has been shown to support a wide variety of two- and three-dimensional cell and tissue culture applications including organoids. Despite widespread use, transport of molecules, cells, and colloidal particles through Matrigel can be limited. These limitations restrict cell growth, viability, and function and limit Matrigel applications. A strategy to improve transport through a hydrogel without modifying the chemistry or composition of the gel is to physically restructure the material into microscopic microgels and then pack them together to form a porous material. These 'granular' hydrogels have been created using a variety of synthetic hydrogels, but granular hydrogels composed of Matrigel have not yet been reported. Here we present a drop-based microfluidics approach for structuring Matrigel into a three-dimensional, mesoporous material composed of packed Matrigel microgels, which we call granular Matrigel. We show that restructuring Matrigel in this manner enhances the transport of colloidal particles and human dendritic cells (DCs) through the gel while providing sufficient mechanical support for culture of human gastric organoids (HGOs) and co-culture of human DCs with HGOs.

Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels.

Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

Rapid parallel generation of a fluorescently barcoded drop library from a microtiter plate using the plate-interfacing parallel encapsulation (PIPE) chip.

In drop-based microfluidics, an aqueous sample is partitioned into drops using individual pump sources that drive water and oil into a drop-making device. Parallelization of drop-making devices is necessary to achieve high-throughput screening of multiple experimental conditions, especially in time-sensitive studies. Here, we present the plate-interfacing parallel encapsulation (PIPE) chip, a microfluidic chip designed to generate 50 to 90 µm diameter drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard 384-well microtiter plate. The PIPE chip is used to generate two types of optically barcoded drop libraries consisting of two-color fluorescent particle combinations: a library of 24 microbead barcodes and a library of 192 quantum dot barcodes. Barcoded combinations in the drop libraries are rapidly measured within a microfluidic device using fluorescence detection and distinct barcoded populations in the fluorescence drop data are identified using DBSCAN data clustering. Signal analysis reveals that particle size defines the source of dominant noise present in the fluorescence intensity distributions of the barcoded drop populations, arising from Poisson loading for microbeads and shot noise for quantum dots. A barcoded population from a drop library is isolated using fluorescence-activated drop sorting, enabling downstream analysis of drop contents. The PIPE chip can improve multiplexed high-throughput assays by enabling simultaneous encapsulation of barcoded samples stored in a microtiter plate and reducing sample preparation time.

Shear-induced disruption of dense nanoemulsion gels.

The structural evolution and rheology of dense nanoemulsion gels, which have been formed by creating strong attractions between slippery nanodroplets, are explored as a function of steady shear rate using rheological small-angle neutron scattering (rheo-SANS). For applied stresses above the yield stress of the gel, the network yields, fracturing into aggregates that break and reform as they tumble and interact in the shear flow. The average aggregate size decreases with increasing shear rate; meanwhile, droplet rearrangements within the clusters, allowed by the slippery nature of the attractive interaction, increase the local density within the aggregates. At the highest shear rates, all clusters disaggregate completely into individual droplets.

Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure.

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.