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.

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.

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.

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.

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.