Featured protocol,
Authors: Marie Beck IversenMarie Beck IversenAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4818 Søren Riis PaludanAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4819 
Christian Kanstrup HolmAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
For correspondence: holm@biomed.au.dkBio-protocol author page: a4820
DOI: https://doi.org/10.21769/BioProtoc.2383.
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| Brief version appeared in Nat Immunol, Feb 2016 |
The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall
et al., 1975; Renis
et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of proliferative cells. Even though this is morphologically different from the human vagina, it closely resembles the endocervical epithelium, which is thought to be the primary site of HSV-2 infection in women (Parr
et al., 1994; Kaushic
et al., 2011). In the protocol presented here, mice are pre-treated with Depo-Provera before intra-vaginal inoculation with HSV-2. The virus replicates in the mucosal epithelium from where it spreads to and replicates in the CNS including the spinal cord, brain stem, cerebrum and cerebellum. Cytokine responses can be detected in vaginal washings using ELISA or in vaginal tissues using qPCR. Further, the recruitment of leukocytes to the vagina can be determined by flow cytometry. The model is suitable for research of both innate and adaptive immunity to HSV-2 infection.
Featured protocol,
Authors: David J. NolanDavid J. NolanAffiliation 1: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Affiliation 2: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4654 Susanna L. LamersAffiliation: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Bio-protocol author page: a4655 Rebecca RoseAffiliation: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Bio-protocol author page: a4656 James J. DollarAffiliation 1: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Affiliation 2: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4657 Marco SalemiAffiliation: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4658 Michael S. McGrathAffiliation 1: Departments of Laboratory Medicine, Pathology, and Medicine, University of California at San Francisco, San Francisco, California, USA
Affiliation 2: The AIDS and Cancer Specimen Resource, University of California at San Francisco, San Francisco, California, USA
For correspondence: MMcGrath@php.ucsf.eduBio-protocol author page: a4659
DOI: https://doi.org/10.21769/BioProtoc.2334.
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| Brief version appeared in J Virol, Sep 2016 |
The current study provides detailed protocols utilized to amplify the complete HIV-1
gp120 and
nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers
et al., 2016a and 2016b; Rose
et al., 2016). This method optimizes protocols from previous publications (Palmer
et al., 2005; Norström
et al., 2012; Lamers
et al., 2015; 2016a and 2016b; Rife
et al., 2016) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.