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.

All the single cells: if you like it then you should put some virus on it.

Across a rich 70-year history, single-cell virology has revealed the impact of host and pathogen heterogeneity during virus infections. Recent technological innovations have enabled higher-resolution analyses of cellular and viral heterogeneity. Furthermore, single-cell analysis has revealed extreme phenotypes and provided additional insights into host-pathogen dynamics. Using a single-cell approach to explore fundamental virology questions, contemporary researchers have contributed to a revival of interest in single-cell virology with increased insights and enthusiasm.

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.

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 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.

Digital droplet RT-LAMP increases speed of SARS-CoV-2 viral RNA detection.

Nucleic acid amplification testing (NAAT) remains one of the most reliable methods for pathogen identification. However, conventional bulk NAATs may not be sufficiently fast or sensitive enough for the detection of clinically-relevant pathogens in point-of-care testing. Here, we have developed a digital droplet RT-LAMP (ddRT-LAMP) assay that rapidly and quantitatively detects the SARS-CoV-2 viral E gene in microfluidic drops. Droplet partitioning using ddRT-LAMP significantly accelerates detection times across a wide range of template concentrations compared to bulk RT-LAMP assays. We discover that a reduction in droplet diameter decreases assay times up to a certain size, upon which surface adsorption of the RT-LAMP polymerase reduces reaction efficiency. Optimization of drop size and polymerase concentration enables rapid, sensitive, and quantitative detection of the SARS-CoV-2 E gene in only 8 min. These results highlight the potential of ddRT-LAMP assays as an excellent platform for quantitative point-of-care testing.

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.

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.

Herpes simplex virus 1 ICP0 phosphorylation site mutants are attenuated for viral replication and impaired for explant-induced reactivation.

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.

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.

Antiviral response mechanisms 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) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection, with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly, SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses 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 can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

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.

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.

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.