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      Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion

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          Abstract

          Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

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          Most cited references45

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          Human rhinoviruses.

          Samantha E Jacobs, Daryl M. Lamson, Kirsten St. George … (2013)
          Human rhinoviruses (HRVs), first discovered in the 1950s, are responsible for more than one-half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work. Advances in molecular methods have enhanced our understanding of the genomic structure of HRV and have led to the characterization of three genetically distinct HRV groups, designated groups A, B, and C, within the genus Enterovirus and the family Picornaviridae. HRVs are traditionally associated with upper respiratory tract infection, otitis media, and sinusitis. In recent years, the increasing implementation of PCR assays for respiratory virus detection in clinical laboratories has facilitated the recognition of HRV as a lower respiratory tract pathogen, particularly in patients with asthma, infants, elderly patients, and immunocompromised hosts. Cultured isolates of HRV remain important for studies of viral characteristics and disease pathogenesis. Indeed, whether the clinical manifestations of HRV are related directly to viral pathogenicity or secondary to the host immune response is the subject of ongoing research. There are currently no approved antiviral therapies for HRVs, and treatment remains primarily supportive. This review provides a comprehensive, up-to-date assessment of the basic virology, pathogenesis, clinical epidemiology, and laboratory features of and treatment and prevention strategies for HRVs.
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            Adherens junction protein nectin-4 (PVRL4) is the epithelial receptor for measles virus

            Michael D. Mühlebach, Mathieu Mateo, Patrick Sinn … (2011)
            Measles (MV) is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths 1,2 . While it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to initially infect macrophages and dendritic cells of the airways using the signaling lymphocytic activation molecule (SLAM, CD150) as receptor 3-6 . These cells then cross the respiratory epithelium and ferry the infection to lymphatic organs where MV replicates vigorously 7 . How and where the virus crosses back into the airways has remained unknown. Based on functional analyses of surface proteins preferentially expressed on virus-permissive epithelial cell lines, we identified nectin-4 8 (poliovirus-receptor-like-4) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains MV entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is down-regulated in infected epithelial cells, including those of macaque tracheas. While other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that MV targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer 9-11 , which has implications for ongoing MV-based clinical trials of oncolysis 12 .
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              Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.

              Cathy L. Mendelsohn, Eckard Wimmer, Vincent Racaniello (1989)
              Restriction of poliovirus replication to a few sites in the infected primate host appears to be controlled by the expression of viral receptors. To learn more about these binding sites and their role in viral tissue tropism, cDNA clones encoding functional poliovirus receptors were isolated. The predicted amino acid sequence reveals that the human poliovirus receptor is an integral membrane protein with the conserved amino acids and domain structure characteristic of members of the immunoglobulin superfamily. Northern hybridization analysis indicates that poliovirus receptor transcripts are expressed in a wide range of human tissues, in contrast to the limited expression of virus binding sites, which suggests that additional factors or modifications of the receptor protein are required to permit poliovirus attachment.
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                Author and article information

                Contributors
                Oliver Schildgen: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 28 September 2015
                Publication date Collection: 2015
                Volume: 10
                Issue: 9
                Electronic Location Identifier: e0138760
                Affiliations
                [001]Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
                Kliniken der Stadt Köln gGmbH, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AY VA RW IHW VP MS UFG. Performed the experiments: AY VA RW. Analyzed the data: AY VA RW VP MS UFG. Contributed reagents/materials/analysis tools: AY VA RW IHW. Wrote the paper: AY VA RW UFG.

                Article
                Publisher ID: PONE-D-15-35163
                DOI: 10.1371/journal.pone.0138760
                PMC ID: 4587671
                PubMed ID: 26413745
                SO-VID: 1102c0ce-1ad7-47ff-819d-a9d73ef19f7e
                Copyright statement: Copyright @ 2015
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                Date received : 10 August 2015
                Date accepted : 24 August 2015
                Page count
                Figures: 7, Tables: 0, Pages: 21
                Funding
                Funding was obtained from the Swiss National Science Foundation (31003A_141222/1 and 310030B_160316 to UFG), the Medical Research and Development project VirX from SystemsX.ch (to UFG), the Novartis Foundation, and the Kanton Zurich. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Custom metadata
                Data Availability The Plaque2.0 software can be downloaded from http://plaque2.github.io/download.html. A user manual and help video can be found at http://plaque2.github.io/. Feature request and bug tracking is available at https://github.com/plaque2/matlab/issues. The source code can be found at https://github.com/plaque2/matlab.

                ScienceOpen disciplines: Uncategorized
                Data availability:
                ScienceOpen disciplines: Uncategorized

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