Emerging SARS-CoV-2 variants expand species tropism to rodents

Abstract Mice are not susceptible to wildtype SARS-CoV-2 infection. Emerging SARS-CoV-2 variants including B.1.1.7, B.1.351, P.1, and P.3 contain mutations in spike, which have been suggested to associate with an increased recognition of mouse ACE2, raising the postulation that they may have evolved to expand species tropism to rodents. Here, we investigated the capacity of B.1.1.7 and other emerging SARS-CoV-2 variants in infecting mouse (Mus musculus) and rats (Rattus norvegicus) under in vitro and in vivo settings. Our results show that B.1.1.7 and P.3, but not B.1 or wildtype SARS-CoV-2, can utilize mouse and rat ACE2 for virus entry in vitro. High infectious virus titers, abundant viral antigen expression, and pathological changes are detected in the nasal turbinate and lung of B.1.1.7-inocluated mice and rats. Together, these results reveal that the current predominant circulating SARS-CoV-2 variant, B.1.1.7, has gained the capability to expand species tropism to rodents.

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Compared Anatomy and Histology between Mus musculus Mice (Swiss) and Rattus norvegicus Rats (Wistar)

10.20944/preprints201907.0306.v1 ◽

2019 ◽

Author(s):

Giorgio Silva-Santana ◽

Fábio Aguiar-Alves ◽

Licínio Esmeraldo Silva ◽

Maria Lúcia Barreto ◽

Jemima Fuentes Ribeiro Silva ◽

...

Keyword(s):

Mus Musculus ◽

Rattus Norvegicus ◽

Experimental Studies ◽

Alternative Methods ◽

Laboratory Animals ◽

Surgical Interventions ◽

Technological Advances ◽

Prophylactic Measures

The evolution of knowledge in biological and medical areas which made possible scientific and technological advances are attributed to anatomical studies, physiology and immunology in animals, contributing to the discovery of prophylactic measures and treatments of diseases that affect humans and animals. Currently, substitution is suggested by alternative methods that do not use laboratory animals, however in vitro methods may never be able to provide similar results to in vivo methods. Mice and rats are the most used animals in experimental researches, the anatomical, histological and genetic differences between species should be carefully evaluated, to better apply the study model and avoid unnecessary waste avoid. This study, aimed at an anatomical comparative and histological relationship between a rat´s and a mouse´s organs, is of great importance in experimental studies. For such, 30 Mus musculus (Swiss) mice and 15 Rattus norvegicus (Wistar) male and heterogenic rats, were used. All animals were kept free of pathogenic microorganisms, with the absence of surgical interventions that could cause anatomical and physiological changes. It was possible to observe significant anatomical and histological differences between spleens, brains, hearts, stomachs and intestines, livers, eyes, lungs and kidneys among species, which will serve as a basis to assist in choosing the most satisfactory research model. Few studies relate specific characteristics among laboratory animals, being restricted to a few veterinary and zoology books. The greater the organic, physiological and anatomical similarities are with the human being, the greater is the applicability of the animals in studies. However, it is not possible to lay down general rules to validate the extrapolation from one species to another.

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Evaluation of cytotoxicity and mutagenicity of the benzodiazepine flunitrazepam in vitro and in vivo

Brazilian Journal of Pharmaceutical Sciences ◽

10.1590/s1984-82502014000200003 ◽

2014 ◽

Vol 50 (2) ◽

pp. 251-256

Author(s):

Igor Vivian de Almeida ◽

Giovana Domingues ◽

Lilian Capelari Soares ◽

Elisângela Düsman ◽

Veronica Elisa Pimenta Vicentini

Keyword(s):

Rattus Norvegicus ◽

Bone Marrow Cells ◽

Micronucleus Test ◽

Term Treatment ◽

Genotoxic Effects ◽

Short Term ◽

Short Term Treatment ◽

Chromosomal Aberration Test

Flunitrazepam (FNZ) is a sedative benzodiazepine prescribed for the short-term treatment of insomnia. However, there are concerns regarding possible carcinogenic or genotoxic effects of this medicine. Thus, the aim of this study was to evaluate the cytotoxic, clastogenic and aneugenic effects of FNZ in hepatoma cells from Rattus norvegicus (HTC) in vitro and in bone marrow cells of Wistar rats in vivo. These effects were examined in vitro following treatment with 0.2, 1.0, 5.0 or 10 μg/mL FNZ using a micronucleus test with a cytokinesis block or in vivo using a chromosomal aberration test following treatment with 7, 15 or 30 μg/mL/kg body weight. The results showed that the benzodiazepine concentrations tested were not cytotoxic, aneugenic or clastogenic. However, considering the adverse effects of using this benzodiazepine, more studies are required.

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The tumor dormant state. Comparison of L5178Y cells used to establish dormancy with those that emerge after its termination.

Journal of Experimental Medicine ◽

10.1084/jem.149.3.745 ◽

1979 ◽

Vol 149 (3) ◽

pp. 745-757 ◽

Cited By ~ 31

Author(s):

K J Weinhold ◽

D A Miller ◽

E F Wheelock

Keyword(s):

Tumor Cells ◽

Antigen Expression ◽

Cell Subpopulation ◽

Effector Cells ◽

Dormant State ◽

Quantitative Absorption ◽

Tumor Outgrowth ◽

In Vivo Growth

The tumor dormant state established in L5178Y immunized and challenged mice is characterized by a prolonged period of clinical normalcy followed by rapid tumor outgrowth. The tumor cells which emerged after termination of the tumor dormant state had abnormal marker chromosomes identical to those in the L5178Y cells used in the original challenge inoculum, indicating that the emergent tumor cells were progeny of the challenge inoculum. Original and emergent L5178Y cells had equivalent in vivo growth rates, when inoculated into normal DBA/2 mice. The emergent L5178Y cells were less susceptible than original cells to in vitro lysis by tumor dormant PC. Original and emergent L5178Y cells expressed common tumor-associated target antigens for cytolytic effector cells. Both modulation and masking of these target antigens were ruled out as mechanisms for decreased susceptibility to cell-mediated cytolysis. Immunofluorescence revealed heterogeneity in tumor-associated antigen expression within both original and emergent cell populations, with a decreased intensity of staining in the emergent population. Both populations were equally susceptible to lysis by alloimmune cells, alloantiserum, and anti-Thy 1.2 serum, but emergent cells were less susceptible to lysis by serum directed against L5178Y TAA. Quantitative absorption revealed that the emergent L5178Y cells expressed eightfold less serologically detectable TAA than the original cells. These findings indicate that the host immune response developing during establishment of the tumor dormant state selects a stable tumor cell subpopulation which expresses decreased amounts of surface tumor-associated target antigens.

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Target-independent regulation of a novel growth associated protein in the visual system of the chicken

Development ◽

10.1242/dev.109.2.395 ◽

1990 ◽

Vol 109 (2) ◽

pp. 395-409 ◽

Cited By ~ 1

Author(s):

B. Schlosshauer ◽

D. Dutting ◽

M. Wild

Keyword(s):

Ganglion Cell ◽

Ganglion Cells ◽

Antigen Expression ◽

Subcellular Fractionation ◽

Axonal Outgrowth ◽

Control Of Gene Expression ◽

Chick Retina ◽

Retinal Axons

Using an immunosuppression technique, the monoclonal antibody 2A1 has been generated specific for a 140 × 10(3) Mr cytoplasmic-membrane-associated protein as shown by subcellular fractionation and Western blot analysis. The antigen is initially confined to perikarya of postmitotic migratory ganglion cells of the embryonic chick retina as revealed by bromodeoxyuridine labeling. During the subsequent period of axon outgrowth, the antigen becomes restricted to ganglion cell axons but disappears during the innervation of the tectum opticum, suggesting a tectal inhibition of antigen expression in retinal axons. To analyse whether the tectum suppresses 2A1-antigen expression, optic nerves of chick embryos were severed to prevent tectal innervation. 2A1-immunoreactivity was determined in deflected axons in comparison to control axons. In addition, retinal axons were grown in vitro on a substratum consisting of alternating stripes of laminin and tectal membranes, in order to investigate whether retinal axons become devoid of the 2A1-antigen once they cross from laminin to tectal membranes. However, neither prevention of target innervation by optic nerve transection in vivo nor exposure of retinal axons to soluble or particulate tectal components in vitro modify 2A1-antigen regulation in ganglion cell axons, suggesting a retina inherent-control of gene expression. Antigen expression is essentially restricted to the period of axonal outgrowth and therefore the 2A1-protein is likely to be involved in processes essential for neurite extension, independent of the synaptic target.

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Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

Journal of Virology ◽

10.1128/jvi.73.4.3326-3337.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 3326-3337 ◽

Cited By ~ 74

Author(s):

Nathalie Arbour ◽

Sophie Ekandé ◽

Geneviève Côté ◽

Claude Lachance ◽

Fanny Chagnon ◽

...

Keyword(s):

Nervous System ◽

Cell Lines ◽

Persistent Infection ◽

Viral Antigen ◽

Infectious Virus ◽

Acute Infection ◽

Viral Rna ◽

Persistent Infections ◽

Virus Progeny

ABSTRACT Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after ∼130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus’s apparent genomic stability.

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Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

Clinical and Vaccine Immunology ◽

10.1128/cvi.00075-15 ◽

2015 ◽

Vol 22 (7) ◽

pp. 726-741 ◽

Cited By ~ 11

Author(s):

Bryan E. Hart ◽

Rose Asrican ◽

So-Yon Lim ◽

Jaimie D. Sixsmith ◽

Regy Lukose ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Antigen Expression ◽

Full Length ◽

Content Type ◽

Immunodeficiency Virus ◽

Vaccine Stability ◽

Hiv Gp120 ◽

Recombinant Bcg

ABSTRACTThe well-established safety profile of the tuberculosis vaccine strain,Mycobacterium bovisbacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCDtransformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging bothin vitroandin vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCDvaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplificationin vitroand following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplificationin vitroand induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCDlots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

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Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Blood ◽

10.1182/blood.v99.6.2084 ◽

2002 ◽

Vol 99 (6) ◽

pp. 2084-2093 ◽

Cited By ~ 101

Author(s):

Alexander D. McLellan ◽

Michaela Kapp ◽

Andreas Eggert ◽

Christian Linden ◽

Ursula Bommhardt ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Antigen Expression ◽

Cell Receptor ◽

In Vitro Assays ◽

Peptide Complexes ◽

Marginal Zones

Abstract Mouse spleen contains CD4+, CD8α+, and CD4−/CD8α− dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule–associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8α+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8α+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)–transgenic T cells. CD8α− or CD8α+DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I–peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8α DCs was responsible for the low allostimulatory capacity of CD8α+ DCs in vitro. Surprisingly, both CD8α+ DCs and CD4−/CD8− DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8α+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8α+ DCs and CD8+ T cells.

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Biocompatibility of Charcoal Hemoperfusion. Effects of Long-Term Treatment on Lymphocyte Characteristics and Function

The International Journal of Artificial Organs ◽

10.1177/039139888600900507 ◽

1986 ◽

Vol 9 (5) ◽

pp. 301-304 ◽

Cited By ~ 3

Author(s):

S. Stefoni ◽

A. Nanni Costa ◽

G. Liviano D'Arcangelo ◽

M. Biavati ◽

S. lannelli ◽

...

Keyword(s):

Antigen Expression ◽

Term Treatment ◽

T Cell Subsets ◽

Long Term Treatment ◽

Circulating Lymphocytes ◽

And Function ◽

Charcoal Hemoperfusion

Biocompatibility of charcoal hemoperfusion was studied in a group of 15 uremic patients, evaluating the effects of long-term treatment on some structural and functional parameters of circulating lymphocytes: in vivo distribution of T-cell subsets; surface T3, T4 and T8 antigen expression, in vivo and in vitro DNA synthesis. A comparative analysis was performed with patients on conventional dialysis using cuprophan membranes.

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3D-microdevice for the in vivo capture of cancer-associated circulating cells.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.e579 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. e579-e579

Author(s):

Hélène Cayron ◽

Alejandro Kayum Jiménez Zenteno ◽

Aurore Esteve ◽

Sylvain Sanson ◽

Christophe Vieu ◽

...

Keyword(s):

Cancer Cells ◽

Expression Profiles ◽

Antigen Expression ◽

Sampling Bias ◽

Additional Treatment ◽

In Vitro Assays ◽

Major Drawback ◽

Direct Laser

e579 Background: Circulating tumor cells (CTCs) are cancer cells that have detached from a tumor and have entered into the blood circulation at a very low concentration (D. Shook, Mech. Dev., Nov 2003). CTCs have a strong prognostic value, as their number has been correlated to overall survival in different metastatic cancers (J. S. de Bono, Clin. Cancer Res., Oct 2008). Considering the rareness of CTCs in blood, capturing them in vitro is very challenging. CTCs being mainly larger and less deformable than most of blood cells, ISET was the first system exploiting their physical traits using a filtration membrane to enrich 10mL blood samples (G. Vona, Am. J. Pathol., Jan 2000). However, placing the trapping system directly within the bloodstream would increase the amount of blood screened and ensure no sampling bias. To our knowledge, the only system developed for in vivo capture of CTCs relies on an immunologic detection targeting CTCs with specific epithelial-cell adhesion molecules (N. Saucedo-Zeni, Int. J. Oncol., Oct 2012). The major drawback of this technique is the selection bias induced, given the strong heterogeneity of antigen expression profiles in CTC population as confirmed by several studies. Methods: Our device combines the advantages of in vivo capture and physical trapping of CTCs. A polymeric 3D net-like microdevice is fabricated using a Direct Laser Writing technique (Nanoscribe) and integrated onto a Nitinol guidewire to be introduced into the basilic vein through a routine 20G catheter. To optimize the design, we conducted simulation studies and in vitro assays using a fluidic platform reproducing in vivo conditions. Results: We succeeded in capturing PC3 human prostate cancer cells from 20 mL healthy donor blood spiked with 1,000 PC3 cells in 2 minutes, demonstrating the capability to capture CTCs in conditions close to those found in vivo, in terms of pressure and flow rate and without any additional treatment or dilution of the blood. Conclusions: This device could facilitate treatment personalization and follow-up. Its versatility should render it transposable to the capture of single or clustered CTCs, derived from all types of cancer and, by extension, to other circulating cellular and molecular biomarkers.

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