An official website of the United States government
Herpes simplex virus type 1 (HSV-1) is a lifelong pathogen characterized by asymptomatic latent infection in the trigeminal ganglia (TG), with periodic outbreaks of cold sores caused by virus reactivation in the TG and subsequent replication in the oral mucosa. While antiviral therapies can provide relief from cold sores, they are unable to eliminate HSV-1. We provide experimental results that highlight non-thermal plasma (NTP) as a new alternative therapy for HSV-1 infection that would resolve cold sores faster and reduce the establishment of latent infection in the TG. Additionally, this study is the first to explore the use of NTP as a therapy that can both treat and prevent human viral infections. The antiviral effect of NTP was investigated using an in vitro model of HSV-1 epithelial infection that involved the application of NTP from two separate devices to cell-free HSV-1, HSV-1-infected cells, and uninfected cells. It was found that NTP reduced the infectivity of cell-free HSV-1, reduced viral replication in HSV-1-infected cells, and diminished the susceptibility of uninfected cells to HSV-1 infection. This triad of antiviral mechanisms of action suggests the potential of NTP as a therapeutic agent effective against HSV-1 infection.
more »
« less
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.
more »
« less
- PAR ID:
- 10539979
- Publisher / Repository:
- Science Journals/AAAS
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 10
- Issue:
- 9
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Kolawole, Abimbola O. (Ed.)ABSTRACT 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.more » « less
-
Abstract Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by theE. colinucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes.more » « less
-
ABSTRACT The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes ( p6.9 , orf76 , orf75 , pp31 , Ac-bro , odv-e25 , and odv-ec27 ) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies ( polh and p10 ) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production ( fp-25k ), acceleration of systemic infection ( v-fgf ), and enhancement of viral movement ( arif-1/orf20 ). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut. IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.more » « less
-
Abstract Viruses persist in nature owing to their extreme genetic heterogeneity and large –population sizes, which enable them to evade host immune defenses, escape anti-viral drugs, and adapt to new hosts. The persistence of viruses is challenging to study because mutations affect multiple virus genes, interactions among genes in their impacts on virus growth are seldom known, and measures of viral fitness have yet to be standardized. To address these challenges, we employed a data-driven computational model of cell infection by a virus. The infection model accounted for the kinetics of viral gene expression, functional gene-gene interactions, genome replication, and allocation of host cellular resources to produce progeny of vesicular stomatitis virus (VSV), a prototype RNA virus. We used this model to computationally probe how interactions among genes carrying up to 11 deleterious mutations affect different measures of virus fitness: single-cycle growth yields and multi-cycle rates of infection spread. Individual mutations were implemented by perturbing biophysical parameters associated with individual gene functions of the wild-type model. Our analysis revealed synergistic epistasis among deleterious mutations in their effects on virus yield; so adverse effects of single deleterious mutations were amplified by interaction. For the same mutations, multi-cycle infection spread indicated weak or negligible epistasis, where single mutations act alone in their effects on infection spread. These results were robust to simulation under high and low host resource environments. Our work highlights how different types and magnitudes of epistasis can arise for genetically identical virus variants, depending on the fitness measure. More broadly, gene-gene interactions can differently affect how viruses grow and spread.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/sciadv.adk9185
