More Like this

1. Rapid and stain-free quantification of viral plaque via lens-free holography and deep learning

   https://doi.org/10.1038/s41551-023-01057-7  

   Liu, Tairan; Li, Yuzhu; Koydemir, Hatice Ceylan; Zhang, Yijie; Yang, Ethan; Eryilmaz, Merve; Wang, Hongda; Li, Jingxi; Bai, Bijie; Ma, Guangdong; et al (June 2023, Nature Biomedical Engineering)

   Abstract A plaque assay—the gold-standard method for measuring the concentration of replication-competent lytic virions—requires staining and usually more than 48 h of runtime. Here we show that lens-free holographic imaging and deep learning can be combined to expedite and automate the assay. The compact imaging device captures phase information label-free at a rate of approximately 0.32 gigapixels per hour per well, covers an area of about 30 × 30 mm²and a 10-fold larger dynamic range of virus concentration than standard assays, and quantifies the infected area and the number of plaque-forming units. For the vesicular stomatitis virus, the automated plaque assay detected the first cell-lysing events caused by viral replication as early as 5 h after incubation, and in less than 20 h it detected plaque-forming units at rates higher than 90% at 100% specificity. Furthermore, it reduced the incubation time of the herpes simplex virus type 1 by about 48 h and that of the encephalomyocarditis virus by about 20 h. The stain-free assay should be amenable for use in virology research, vaccine development and clinical diagnosis. 

   more » « less
2. Multicellular spatial model of RNA virus replication and interferon responses reveals factors controlling plaque growth dynamics

   https://doi.org/10.1371/journal.pcbi.1008874  

   Aponte-Serrano, Josua O.; Weaver, Jordan J.; Sego, T. J.; Glazier, James A.; Shoemaker, Jason E. (October 2021, PLOS Computational Biology)

   Kosakovsky Pond, Sergei L. (Ed.)

   Respiratory viruses present major public health challenges, as evidenced by the 1918 Spanish Flu, the 1957 H2N2, 1968 H3N2, and 2009 H1N1 influenza pandemics, and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestations at the cell and tissue levels is vital to understanding the mechanisms of immunopathology and to developing strain-independent treatments. Here, we present a novel spatialized multicellular computational model of RNA virus infection and the type-I interferon-mediated antiviral response that it induces within lung epithelial cells. The model is built using the CompuCell3D multicellular simulation environment and is parameterized using data from influenza virus-infected cell cultures. Consistent with experimental observations, it exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. The model suggests that modifying the activity of signaling molecules in the JAK/STAT pathway or altering the ratio of the diffusion lengths of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of plaque growth arrest on diffusion lengths highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro measurement of these diffusion coefficients. Sensitivity analyses under conditions leading to continuous or arrested plaque growth found that plaque growth is more sensitive to variations of most parameters and more likely to have identifiable model parameters when conditions lead to plaque arrest. This result suggests that cytokine assay measurements may be most informative under conditions leading to arrested plaque growth. The model is easy to extend to include SARS-CoV-2-specific mechanisms or to use as a component in models linking epithelial cell signaling to systemic immune models. 

   more » « less
3. Viral tag and grow: a scalable approach to capture and characterize infectious virus–host pairs

   https://doi.org/10.1038/s43705-022-00093-9  

   Jang, Ho Bin; Chittick, Lauren; Li, Yueh-Fen; Zablocki, Olivier; Sanderson, Courtney M.; Carrillo, Alfonso; van den Engh, Ger; Sullivan, Matthew B. (February 2022, ISME Communications)

   Abstract Viral metagenomics (viromics) has reshaped our understanding of DNA viral diversity, ecology, and evolution across Earth’s ecosystems. However, viromics now needs approaches to link newly discovered viruses to their host cells and characterize them at scale. This study adapts one such method, sequencing-enabled viral tagging (VT), to establish “Viral Tag and Grow” (VT + Grow) to rapidly capture and characterize viruses that infect a cultivated target bacterium, Pseudoalteromonas. First, baseline cytometric and microscopy data improved understanding of how infection conditions and host physiology impact populations in VT flow cytograms. Next, we extensively evaluated “and grow” capability to assess where VT signals reflect adsorption alone or wholly successful infections that lead to lysis. Third, we applied VT + Grow to a clonal virus stock, which, coupled to traditional plaque assays, revealed significant variability in burst size—findings that hint at a viral “individuality” parallel to the microbial phenotypic heterogeneity literature. Finally, we established a live protocol for public comment and improvement via protocols.io to maximally empower the research community. Together these efforts provide a robust foundation for VT researchers, and establish VT + Grow as a promising scalable technology to capture and characterize viruses from mixed community source samples that infect cultivable bacteria. 

   more » « less
4. Chloroviruses

   https://doi.org/10.3390/v12010020  

   Van Etten, James L.; Agarkova, Irina V.; Dunigan, David D. (January 2020, Viruses)

   Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation. 

   more » « less
5. The effect of silicon on the kinetics of rice root iron plaque formation

   https://doi.org/10.1007/s11104-022-05414-4  

   Limmer, Matt A.; Thomas, John; Seyfferth, Angelia L. (April 2022, Plant and Soil)

   Abstract PurposeAquatic plants, including rice, develop iron (Fe) plaques on their roots due to radial oxygen loss (ROL), and these plaques accumulate both beneficial and toxic elements. Silicon is an important nutrient for rice and both accumulates in Fe plaque and can affect ROL. How these plaques form over time and how Si affects this process remains unclear. MethodsRice was grown in a pot study with 4 levels of added Si. Root Fe plaque formation was monitored weekly using vinyl films placed between the pot and soil. Plants were grown to maturity and then ratooned to also examine the formation of Fe plaque during the ratoon crop. ResultsIron plaque formation increased exponentially during the vegetative phase, peaked at the booting phase, then decreased exponentially – a pattern that repeated in the ratoon crop. While the highest Si treatment led to an earlier onset of Fe plaque formation, increasing Si decreased the amount of Fe plaque at harvest, resulting in a minimal net effect. ConclusionsThe kinetics of Fe plaque formation are dependent on rice growth stage, which may affect whether the Fe plaque is a source or sink of elements such as phosphorous and arsenic. 

   more » « less