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University of Rochester School of Medicine and Dentistry

9 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA
Luis Martínez-Sobrido 1, Adolfo García-Sastre 2
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

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Immunology and Infection

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells
Benson Y.H. Cheng *1, Emilio Ortiz-Riaño *1, Juan Carlos de la Torre 2, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Immunology and Microbial Sciences, The Scripps Research Institute

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

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Immunology and Infection

Rescue of Recombinant Newcastle Disease Virus from cDNA
Juan Ayllon 1,2, Adolfo García-Sastre 1,2,3, Luis Martínez-Sobrido 4
1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, 3Department of Medicine, Icahn School of Medicine at Mount Sinai, 4Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

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Developmental Biology

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration
Michael A. Trembley 1,3, Lissette S. Velasquez 1, Eric M. Small 1,2,3
1Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 2Department of Medicine, University of Rochester School of Medicine and Dentistry, 3Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

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JoVE Journal

Influenza A Virus Studies in a Mouse Model of Infection
Laura Rodriguez 1, Aitor Nogales 1, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

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Immunology and Infection

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone
Ginés Ávila-Pérez 1, Jun-Gyu Park 1, Aitor Nogales 1, Fernando Almazán 2, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

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Immunology and Infection

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection
Kevin Chiem 1, Javier Rangel-Moreno 2, Aitor Nogales 1,3, Luis Martinez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Division of Allergy/Immunology and Rheumatology, Department of Medicine, University of Rochester, 3Center for Animal Health Research, INIA-CISA

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

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Immunology and Infection

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2
Desarey Morales Vasquez 1, Kevin Chiem 1, Jesus Silvas 1, Jun-Gyu Park 1, Chengjin Ye 1, Luis Martínez-Sobrido 1
1Texas Biomedical Research Institute

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

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Biology

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid
Muhammad Ummear Raza 1, Kavaljit H. Chhabra 1
1Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Rochester School of Medicine and Dentistry

This article describes a detailed protocol to increase glucose concentration in the cerebrospinal fluid (CSF) of mice. This approach can be useful for studying the effects of high CSF glucose on neurodegeneration, cognition, and peripheral glucose metabolism in mice.

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