scholarly journals Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yue Zheng ◽  
Erin T. Larragoite ◽  
Elizabeth S. C. P. Williams ◽  
Juan Lama ◽  
Isabel Cisneros ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Neutralizing Antibodies ◽  
Influenza Infection ◽  
Leukemia Virus ◽  
Neutralization Assay ◽  
Spike Glycoprotein ◽  
Serum Samples ◽  
Neutralizing Activity ◽  
Level 2 ◽  
Murine Leukemia

Abstract Background Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. Results 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). Conclusions SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

scholarly journals Neutralization Assay with SARS-CoV-1 and SARS-CoV-2 Spike Pseudotyped Murine Leukemia Virions

2020 ◽  
Author(s):  
Yue Zheng ◽  
Erin T. Larragoite ◽  
Juan Lama ◽  
Isabel Cisneros ◽  
Julio C. Delgado ◽  
...  
Keyword(s):  
Immune Responses ◽  
Murine Leukemia Virus ◽  
Neutralizing Antibodies ◽  
Leukemia Virus ◽  
Firefly Luciferase ◽  
Neutralization Assay ◽  
Viral Diseases ◽  
Virus Capsids ◽  
Level 2 ◽  
Murine Leukemia

AbstractAntibody neutralization is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal MLV genome encoding firefly luciferase. These pseudovirions provide a practical means of assessing immune responses under laboratory conditions consistent with biocontainment level 2.

scholarly journals Prevention of Contamination by Xenotropic Murine Leukemia Virus-Related Virus: Susceptibility to Alcohol-Based Disinfectants and Environmental Stability

2014 ◽  
Vol 80 (8) ◽  
pp. 2617-2622
Author(s):  
David Palesch ◽  
Mohammad Khalid ◽  
Christina M. Stürzel ◽  
Jan Münch
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Environmental Stability ◽  
Xenotropic Murine Leukemia ◽  
Related Virus ◽  
Different Temperatures ◽  
Level 2 ◽  
Biosafety Level ◽  
Murine Leukemia ◽  
Biosafety Level 2

ABSTRACTXenotropic murine leukemia virus-related virus (XMRV) represents a novel γ-retrovirus that is capable of infecting human cells and has been classified as a biosafety level 2 (BSL-2) organism. Hence, XMRV represents a potential risk for personnel in laboratories worldwide. Here, we measured the stability of XMRV and its susceptibility to alcohol-based disinfectants. To this end, we exposed an infectious XMRV reporter virus encoding a secretable luciferase to different temperatures, pH values, and disinfectants and infected XMRV-permissive Raji B cells to measure residual viral infectivity. We found that 1 min treatment of XMRV particles at 60°C is sufficient to reduce infectivity by 99.9%. XMRV infectivity was maximal at a neutral pH but was reduced by 86% at pH 4 and 99.9% at pH 10. The common hand and surface disinfectants ethanol and isopropanol as well as the cell fixation reagent paraformaldehyde abrogated XMRV infectivity entirely, as indicated by a reduction of infectivity exceeding 99.99%. Our findings provide evidence of specific means to inactivate XMRV. Their application will help to prevent unintended XMRV contamination of cell cultures in laboratories and minimize the risk for laboratory personnel and health care workers to become infected with this biosafety level 2 organism.

scholarly journals Distinct Mechanisms of Neutralization by Monoclonal Antibodies Specific for Sites in the N-Terminal or C-Terminal Domain of Murine Leukemia Virus SU

2003 ◽  
Vol 77 (7) ◽  
pp. 3993-4003 ◽  
Author(s):  
Michael Dominic Burkhart ◽  
Samuel C. Kayman ◽  
Yuxian He ◽  
Abraham Pinter
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Murine Leukemia Virus ◽  
Neutralizing Antibodies ◽  
Leukemia Virus ◽  
Binding Pocket ◽  
Protein Subunit ◽  
Viral Neutralization ◽  
Terminal Domain ◽  
Murine Leukemia

ABSTRACT The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.

scholarly journals Neutralizing activity and cellular immune responses induced in mice after immunization with apoptotic HIV-1/murine leukemia virus infected cells

Vaccine ◽  
2009 ◽  
Vol 27 (46) ◽  
pp. 6424-6431 ◽  
Author(s):  
Jorma Hinkula ◽  
Lilian Walther-Jallow ◽  
Anna Laurén ◽  
Barbro Mäkitalo ◽  
Monica Öberg ◽  
...  
Keyword(s):  
Immune Responses ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Neutralizing Activity ◽  
Cellular Immune Responses ◽  
Infected Cells ◽  
Cellular Immune ◽  
Hiv 1 ◽  
Murine Leukemia

scholarly journals Stoichiometry of Murine Leukemia Virus Envelope Protein-Mediated Fusion and Its Neutralization

2006 ◽  
Vol 80 (24) ◽  
pp. 11982-11990 ◽  
Author(s):  
Wu Ou ◽  
Jonathan Silver
Keyword(s):  
Murine Leukemia Virus ◽  
Neutralizing Antibodies ◽  
Fundamental Problem ◽  
Leukemia Virus ◽  
Point Mutations ◽  
Neutralizing Antibody ◽  
Virus Envelope ◽  
Antibody Molecule ◽  
Virus Envelope Protein ◽  
Murine Leukemia

ABSTRACT Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.

Recent Studies on a Murine Leukemia Virus Grown in Tissue Culture

1967 ◽  
Vol 25 ◽  
pp. 106-107
Author(s):  
L. Z. de Tkaczevski ◽  
E. de Harven ◽  
C. Friend
Keyword(s):  
Tissue Culture ◽  
Solid Tumors ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Supernatant Fluid ◽  
Virus Particles ◽  
Friend Leukemia ◽  
Type C ◽  
Leukemia Viruses ◽  
Murine Leukemia

Despite extensive studies, the correlation between the morphology and pathogenicity of murine leukemia viruses (MLV) has not yet been clarified. The virus particles found in the plasma of leukemic mice belong to 2 distinct groups, 1 or 2% of them being enveloped A particles and the vast majority being of type C. It is generally believed that these 2 types of particles represent different phases in the development of the same virus. Particles of type A have been thought to be an earlier form of type C particles. One of the tissue culture lines established from Friend leukemia solid tumors has provided the material for the present study. The supernatant fluid of the line designated C-1A contains an almost pure population of A particles as illustrated in Figure 1. The ratio is, therefore, the reverse of what is unvariably observed in the plasma of leukemic mice where C particles predominate.

Immunoferritin Labelling of Murine Leukemia Virus Antigens in thin Sections

1980 ◽  
Vol 38 ◽  
pp. 508-509
Author(s):  
Ray A. Weigand ◽  
Gregory C. Varjabedian
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Intracellular Localization ◽  
Uranyl Acetate ◽  
Antibody Technique ◽  
Thin Sections ◽  
Tumor Virus ◽  
Labelling Procedure ◽  
Unlabelled Antibody ◽  
Murine Leukemia

We previously described the intracellular localization of murine mammary tumor virus (MuMTV) p28 protein in thin sections (1). In that study, MuMTV containing cells fixed in 3% paraformaldehyde plus 0.05% glutaraldehyde were labelled after thin sectioning using ferritin-antiferritin in an unlabelled antibody technique. We now describe the labelling of murine leukemia virus (MuLV) particles using the unlabelled antibody technique coupled to ferritin-Fab antiferritin. Cultures of R-MuLV in NIH/3T3 cells were grown to 90% confluence (2), fixed with 2% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M cacodylate at pH 7.2, postfixed with buffered 17 OsO4, dehydrated with a series of etha-nols, and embedded in Epon. Thin sections were collected on nickel grids, incubated in 107 H2O2, rinsed in HEPES buffered saline, and subjected to the immunoferritin labelling procedure. The procedure included preincubation in 27 egg albumin, a four hour incubation in goat antisera against purified gp69/71 of MuLV (3) (primary antibody), incubation in F(ab’)2 fragments of rabbit antisera to goat IgG (secondary antibody), incubation in apoferritin, incubation in ferritin-Fab ferritin, and a brief fixation with 2% glutaraldehyde. The sections were stained with uranyl acetate and examined in a Siemens IA electron microscope at an accelerating voltage of 60 KV.

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