Protocols in Current Issue
Protocols in Past Issues
0 Q&A 2065 Views Dec 20, 2021

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate. However, recent data suggests poxvirus genomes are not repaired well following Cas9 cleavage. As a result, CRISPR/Cas9 is inefficient as an editing tool, but very effective as a programmable selection agent. Here, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses. This is also the first method that allows for isolation of new vaccinia viruses in less than a fortnight, without the need to incorporate a marker gene or manipulation of large poxvirus genomes in vitro and reactivation with helper viruses. Whilst this protocol describes applications for laboratory strains of vaccinia virus, it should be readily adaptable to other poxviruses.

Graphic abstract:

Pipeline for Cas9 selection of recombinant poxviruses.

0 Q&A 4541 Views Apr 20, 2019
Kaposi’s sarcoma (KS) herpesvirus (KSHV) is a virus that causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Many host signaling cascades co-opted by KSHV including PI3K/AKT/mTORC, NFkB and Notch are critical for cell-specific mechanisms of transformation and their identification is paving the way to therapeutic target discovery. Analysis of the molecular KS signature common to human KS tumors and our mouse KS-like tumors showed consistent expression of KS markers VEGF and PDGF receptors with upregulation of other angiogenesis ligands and their receptors in vivo. This points to the autocrine and paracrine activation of various receptor tyrosine kinase (RTK) signaling axes. Hereby we describe a protocol to screen for activated receptor tyrosine kinase of KSHV-induced KS-like mouse tumors using a Mouse Phospho-RTK Array Kit and its validation by RTK western blots. We showed that this method can be successfully used to rank the tyrosine kinase receptors most activated in tumors in an unbiased manner. This allowed us to identify PDGFRA as an oncogenic driver and therapeutic target in AIDS-KS.
0 Q&A 5788 Views Oct 20, 2018
Influenza A virus is a member of orthomyxoviridae family causing wide-spread infections in human respiratory tract. Mouse infection model is widely used in antiviral research and pathogenesis study against influenza A virus. Here, we report a protocol in infected mice with different virus doses and strains to explore how an inhibitor of lysine-specific demethylase (LSD1) impacts disease progression.
0 Q&A 5102 Views Aug 5, 2018
Hepatitis C virus (HCV) spread involves two distinct entry pathways: cell-free transmission and cell-to-cell transmission. Cell-to-cell transmission is not only an efficient way for viruses to spread but also an effective method for escaping neutralizing antibodies. We adapted the viral infection-activated split-intein-mediated reporter system (VISI) and developed a straightforward model for Live-cell monitoring of HCV cell-to-cell transmission ex-vivo: co-culture of HCV infected donor cells (red signal) with uninfected recipient cells (green signal) and elimination of the cell-free transmission by adding potent neutralizing antibody AR3A in the supernatant. With this model, the efficiency of cell-to-cell transmission can be evaluated by counting the number of foci designated by the green signal of recipient cells.
0 Q&A 5528 Views May 5, 2018
Human norovirus is the most common cause of acute gastroenteritis worldwide, resulting in estimated mortality of ~210,000 each year, of whom most are children under the age of five. However, norovirus can infect people of all age groups. There is a risk of prolonged infection in children, the elderly and patients who are immunocompromised. To study the inhibition of persistent norovirus replication by small molecule antivirals in vivo, we used a murine norovirus CR6 strain (MNV.CR6). We demonstrated earlier that efficient small molecules can reduce viral shedding in the stool of infected mice. Here we present how to generate the MNV.CR6 virus stock, infect type I and II interferon receptor knockout AG129 mice via oral gavage, administer antivirals to mice, and quantify viral genome copies in the stool of these mice.
0 Q&A 7711 Views Nov 20, 2017
Over the past 15 years, the free-living nematode, Caenorhabditis elegans has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in C. elegans. These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms. Here we report protocols for the infection of C. elegans with a non-natural viral pathogen, vesicular stomatitis virus (VSV) through microinjection. We also describe how recombinant VSV strains encoding fluorescent or luciferase reporter genes can be used in conjunction with simple fluorescence-, survival-, and luminescence-based assays to identify host genetic backgrounds with differential susceptibilities to virus infection.
0 Q&A 9159 Views Jul 5, 2017
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.
0 Q&A 9380 Views Dec 5, 2016
Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated with secondary heterotypic infections through a phenomenon that is described as antibody-dependent enhancement of infection (ADE). There are an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly. At present, however, there are no approved antiviral drugs against DENV infection. The lack of a suitable animal model has hampered the evaluation of novel antiviral candidates for DENV infection. Since DENV poorly establishes infection in immunocompetent mice, AG129 mice (lacking type I and II IFN [interferon] receptors) and mouse-adapted DENV2 strains have been applied to dengue animal models that enable to reproduce several of the major pathologies of human infection. Recently, we developed new mouse models with clinical isolates DENV1 and DENV2 that would be useful for drug testing and dengue pathogenesis studies (Watanabe et al., 2016). Here we describe the details to establish dengue mouse models of clinical isolates; from in vitro preparation of the materials to in vivo virus infection. Of note, since infectivity of DENV in mice differs among virus strains, not all clinical isolates can induce severe dengue.
0 Q&A 10087 Views May 20, 2016
Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated with the development and exacerbation of chronic lung conditions including asthma, and it is a major cause of hospitalizations in infants. Because of its clinical relevance, experimental animal models to study RSV in vivo are needed. The most common and accessible animal model in research laboratories is the mouse. However, commonly use RSV strains poorly establish infection in mice and thus titration of the virus from mouse lungs to confirm infection is not sensitive enough to detect early viral infection. Here we discuss in detail how to infect BALB/c mice with RSV and how to detect RSV genomes in the lung using reverse transcription quantitative PCR (RT-qPCR). This method allows detection of viral genomes as early as day 1 post-infection (shown in Figure 2), whereas traditional TCID50 fails to detect significant virus until after day 2 post-infection. Of note, despite of higher sensitivity, genome RT-qPCR only shows the production of viral genomes and thus positive results for this assay are not proof of production of infectious viral particles.

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