scholarly journals Vaccinia virus semaphorin A39R is a 50–55 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model

2001 ◽  
Vol 82 (9) ◽  
pp. 2083-2093 ◽  
Author(s):  
Jeremy D. Gardner ◽  
David C. Tscharke ◽  
Patrick C. Reading ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Cytokine Production ◽  
Mouse Skin ◽  
Skin Lesions ◽  
Lesion Size ◽  
Binding Properties ◽  
Amino Acid Similarity ◽  
Virus Growth ◽  
Recombinant Viruses

Vaccinia virus (VV) protein A39R has amino acid similarity to the extracellular domain of a glycosylphosphatidylinositol-linked cell surface semaphorin (SEMA7A/CDw108) that has an immunological expression profile and binding properties, thereby implicating A39R as an immunomodulator. Previously, a closely related A39R protein expressed by ectromelia virus was shown to induce cytokine production and up-regulate ICAM-1 expression in mouse monocytes in vitro. In this study, we show that the A39R gene of VV strain Copenhagen (COP) encodes a 50–55 kDa secreted glycoprotein and is expressed late during infection. The A39R protein was secreted by eight of 15 strains of VV, but not by strain Western Reserve (WR). To analyse the VV A39R function, several recombinant viruses were made, including an A39R deletion mutant of VV COP and a WR mutant containing the A39R sequence from COP. Loss of the gene from COP did not affect virus growth in vitro, or VV virulence in a mouse intranasal model, and had only a slight effect on lesion size in an intradermal model. In contrast, expression of COP A39R by VV WR was associated with an increase in the severity and persistence of skin lesions after intradermal infection of mice. Finally, a histological examination of mouse skin infected with recombinant viruses suggested that A39R has direct or indirect pro-inflammatory properties.

scholarly journals Effect of Topical Liposomes Containing Paromomycin Sulfate in the Course of Leishmania major Infection in Susceptible BALB/c Mice

2009 ◽  
Vol 53 (6) ◽  
pp. 2259-2265 ◽  
Author(s):  
Mahmoud R. Jaafari ◽  
Neda Bavarsad ◽  
Bibi Sedigheh Fazly Bazzaz ◽  
Afshin Samiei ◽  
Dina Soroush ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Mouse Skin ◽  
Parasite Burden ◽  
Lesion Size ◽  
Control Group ◽  
In Vitro Permeation ◽  
Significant Difference ◽  
Pm 10 ◽  
Franz Diffusion Cells

ABSTRACT The aim of this study was to evaluate the antileishmanial effects of topical liposomal paromomycin sulfate (PM) in Leishmania major-infected BALB/c mice. Liposomes containing 10 or 15% PM (Lip-PM-10 and Lip-PM-15, respectively) were prepared by the fusion method and were characterized for their size and encapsulation efficiency. The penetration of PM from the liposomal PM formulations (LPMFs) through and into skin was evaluated in vitro with Franz diffusion cells fitted with mouse skin at 37°C for 8 h. The in vitro permeation data showed that almost 15% of the LPMFs applied penetrated the mouse skin, and the amount retained in the skin was about 60% for both formulations. The 50% effective doses of Lip-PM-10 and Lip-PM-15 against L. major promastigotes in culture were 65.32 and 59.73 μg/ml, respectively, and those against L. major amastigotes in macrophages were 24.64 and 26.44 μg/ml, respectively. Lip-PM-10 or Lip-PM-15 was used topically twice a day for 4 weeks to treat L. major lesions on BALB/c mice, and the results showed a significantly (P < 0.001) smaller lesion size in the mice in the treated groups than in the mice in the control group, which received either empty liposomes or phosphate-buffered saline (PBS). Eight weeks after the beginning of the treatment, every mouse treated with LPMFs was completely cured. The spleen parasite burden was significantly (P < 0.001) lower in mice treated with Lip-PM-10 or Lip-PM-15 than in mice treated with PBS or control liposomes, but no significant difference was seen between the two groups treated with either Lip-PM-10 or Lip-PM-15. The results suggest that topical liposomal PM may be useful for the treatment of cutaneous leishmaniasis.

scholarly journals Reverse Genetic Generation of Recombinant Zaire Ebola Viruses Containing Disrupted IRF-3 Inhibitory Domains Results in Attenuated Virus Growth In Vitro and Higher Levels of IRF-3 Activation without Inhibiting Viral Transcription or Replication

2006 ◽  
Vol 80 (13) ◽  
pp. 6430-6440 ◽  
Author(s):  
Amy L. Hartman ◽  
Jason E. Dover ◽  
Jonathan S. Towner ◽  
Stuart T. Nichol
Keyword(s):  
Reverse Genetics ◽  
Ebola Virus ◽  
Viral Transcription ◽  
Type I ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Inhibitory Function ◽  
C Terminus ◽  
Inhibitory Domain

ABSTRACT The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-β production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.

scholarly journals Cidofovir Resistance in Vaccinia Virus Is Linked to Diminished Virulencein Mice

2006 ◽  
Vol 80 (19) ◽  
pp. 9391-9401 ◽  
Author(s):  
Graciela Andrei ◽  
Don B. Gammon ◽  
Pierre Fiten ◽  
Erik De Clercq ◽  
Ghislain Opdenakker ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Vaccinia Virus ◽  
Dna Polymerase ◽  
In Vitro Selection ◽  
Cross Resistance ◽  
Gene Encoding ◽  
Recombinant Viruses ◽  
Substitution Mutations

ABSTRACT Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3′-5′ exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

scholarly journals Vaccinia virus gene F3L encodes an intracellular protein that affects the innate immune response

2007 ◽  
Vol 88 (7) ◽  
pp. 1917-1921 ◽  
Author(s):  
Graham C. Froggatt ◽  
Geoffrey L. Smith ◽  
Philippa M. Beard
Keyword(s):  
Immune Response ◽  
Vaccinia Virus ◽  
Innate Immune Response ◽  
Cytopathic Effect ◽  
Innate Immune ◽  
Intracellular Protein ◽  
Virus Growth ◽  
Plaque Phenotype

The Vaccinia virus BTB/kelch protein F3 has been characterized and its effects on virus replication in vitro and virus virulence in vivo have been determined. The loss of the F3L gene had no effect on virus growth, plaque phenotype or cytopathic effect in cell culture under the conditions tested. However, the virulence of a virus lacking F3L in an intradermal model was reduced compared with controls, and this was demonstrated by a significantly smaller lesion and alterations to the innate immune response to infection. The predicted molecular mass of the F3 protein is 56 kDa; however, immunoblotting of infected cell lysates using an antibody directed against recombinant F3 revealed two proteins of estimated sizes 37 and 25 kDa.

scholarly journals The therapeutic effects of larval excretion/secretion of Lucilia sericata on Leishmania major under in vitro and in vivo conditions

2021 ◽  
Author(s):  
Jila Sherafati ◽  
Mohammad Saaid Dayer ◽  
Fatemeh Ghaffarifar
Keyword(s):  
Leishmania Major ◽  
Skin Lesions ◽  
Lesion Size ◽  
Negative Control ◽  
Lucilia Sericata ◽  
Definitive Treatment ◽  
Therapeutic Effects ◽  
Model Animal

Abstract Background Leishmaniasis is a neglected infectious disease caused by a kinetoplastid protozoan. The disease generally manifests as characteristic skin lesions. Due to the lack of definitive treatment and drugs without side effects, many studies have focused on natural compounds as promising drugs for its treatment. This study aimed to evaluate the effects of larval excretion/secretion products (ES) of Lucilia sericata in crude and fractionated forms on Leishmania parasites under both in vitro and in vivo conditions. Methods In vitro experiments involved evaluation of ES products on both promastigotes and amastigotes inside infected macrophages, whereas in vivo experiments included comparative treatments of Leishmanial lesions of mice using Eucerin-formulated ES products and glucantime. Results The IC50 values were 38.7 µg/ml, 47.6 µg/ml, 63.3 µg/ml, and 29.1 µg/ml for crude ES, over 10 kDa ES-fraction, under 10 kDa ES-fraction, and glucantime respectively. Significant differences were observed between viability percentages of promastigotes treated with crude ES and its fractions compared to negative control (p < 0.0001). Crude ES was more effective on amastigote than other two ES fractions at 300 µg/ml concentration. Macroscopic measurement of lesion sizes revealed that the reduction of lesion size in mice treated with crude ES followed quicker cascades of healing than in those treated with glucantime and fractionated ES. Conclusion The present study showed that larval ES of Lucilia sericata in both crude and fractionated forms are effective on both intracellular and extracellular forms of L. major. It also provided evidence that the larval ES exerts both topical and systemic therapeutic effects on leishmanial lesions of the model animal.

scholarly journals Topical Treatment for Cutaneous Leishmaniasis: Dermato-Pharmacokinetic Lead Optimization of Benzoxaboroles

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Katrien Van Bocxlaer ◽  
Eric Gaukel ◽  
Deirdre Hauser ◽  
Seong Hee Park ◽  
Sara Schock ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Leishmania Major ◽  
Skin Permeation ◽  
Mouse Skin ◽  
Topical Administration ◽  
Lesion Size ◽  
Drug Formulation ◽  
Content Type

ABSTRACTCutaneous leishmaniasis (CL) is caused by several species of the protozoan parasiteLeishmania, affecting an estimated 10 million people worldwide. Previously reported strategies for the development of topical CL treatments have focused primarily on drug permeation and formulation optimization as the means to increase treatment efficacy. Our approach aims to identify compounds with antileishmanial activity and properties consistent with topical administration. Of the test compounds, five benzoxaboroles showed potent activity (50% effective concentration [EC50] < 5 μM) against intracellular amastigotes of at least oneLeishmaniaspecies and acceptable activity (20 μM < EC50< 30 μM) against two more species. Benzoxaborole compounds were further prioritized on the basis of thein vitroevaluation of progression criteria related to skin permeation, such as the partition coefficient and solubility. An MDCKII-hMDR1 cell assay showed overall good permeability and no significant interaction with the P-glycoprotein transporter for all substrates except LSH002 and LSH031. The benzoxaboroles were degraded, to some extent, by skin enzymes but had stability superior to that ofpara-hydroxybenzoate compounds, which are known skin esterase substrates. Evaluation of permeation through reconstructed human epidermis showed LSH002 to be the most permeant, followed by LSH003 and LSH001. Skin disposition studies following finite drug formulation application to mouse skin demonstrated the highest permeation for LSH001, followed by LSH003 and LSH002, with a significantly larger amount of LSH001 than the other compounds being retained in skin. Finally, the efficacy of the leads (LSH001, LSH002, and LSH003) againstLeishmania majorwas testedin vivo. LSH001 suppressed lesion growth upon topical application, and LSH003 reduced the lesion size following oral administration.

scholarly journals Activity of Amphotericin B-Loaded Chitosan Nanoparticles against Experimental Cutaneous Leishmaniasis

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 4002
Author(s):  
Alaa Riezk ◽  
Katrien Van Bocxlaer ◽  
Vanessa Yardley ◽  
Sudaxshina Murdan ◽  
Simon L. Croft
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Amphotericin B ◽  
Leishmania Major ◽  
Chitosan Nanoparticles ◽  
Mouse Skin ◽  
Parasite Load ◽  
Lesion Size ◽  
Franz Diffusion Cells ◽  
Positively Charged

Chitosan nanoparticles have gained attention as drug delivery systems (DDS) in the medical field as they are both biodegradable and biocompatible with reported antimicrobial and anti-leishmanial activities. We investigated the application of chitosan nanoparticles as a DDS for the treatment of cutaneous leishmaniasis (CL) by preparing two types of chitosan nanoparticles: positively charged with tripolyphosphate sodium (TPP) and negatively charged with dextran sulphate. Amphotericin B (AmB) was incorporated into these nanoparticles. Both types of AmB-loaded nanoparticles demonstrated in vitro activity against Leishmania major intracellular amastigotes, with similar activity to unencapsulated AmB, but with a significant lower toxicity to KB-cells and red blood cells. In murine models of CL caused by L. major, intravenous administration of AmB-loaded chitosan-TPP nanoparticles (Size = 69 ± 8 nm, Zeta potential = 25.5 ± 1 mV, 5 mg/kg/for 10 days on alternate days) showed a significantly higher efficacy than AmBisome® (10 mg/kg/for 10 days on alternate days) in terms of reduction of lesion size and parasite load (measured by both bioluminescence and qPCR). Poor drug permeation into and through mouse skin, using Franz diffusion cells, showed that AmB-loaded chitosan nanoparticles are not appropriate candidates for topical treatment of CL.

scholarly journals Mechanism of Antiviral Drug Resistance of Vaccinia Virus: Identification of Residues in the Viral DNA Polymerase Conferring Differential Resistance to Antipoxvirus Drugs

2008 ◽  
Vol 82 (24) ◽  
pp. 12520-12534 ◽  
Author(s):  
Don B. Gammon ◽  
Robert Snoeck ◽  
Pierre Fiten ◽  
Marcela Krečmerová ◽  
Antonín Holý ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Vaccinia Virus ◽  
Dna Polymerase ◽  
Antiviral Drug ◽  
Growth Defect ◽  
Variable Degree ◽  
Virus Growth ◽  
Virus Identification ◽  
Selection For

ABSTRACT The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a ∼15-fold-increased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.

scholarly journals Bi-reporter vaccinia virus for tracking viral infections in vitro and in vivo

2021 ◽  
Author(s):  
Kevin Chiem ◽  
María Lorenzo ◽  
Javier Rangel-Moreno ◽  
Maria de la Luz Garcia-Hernandez ◽  
Jun-Gyu Park ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Viral Infections ◽  
Viral Vector ◽  
Ex Vivo ◽  
Reporter Genes ◽  
Luciferase Reporter ◽  
Recombinant Viruses ◽  
Antibody Staining

Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. In vitro, these bi-reporter expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. In vivo, rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using in vivo imaging systems (IVIS) and ex vivo in the lungs from infected mice. We used these bi-reporter expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections in vitro and in vivo, including host-pathogen interactions and dynamics or tropism of viral infections. Moreover, they represent an excellent approach for the discovery of new prophylactics and/or therapeutics for the treatment of poxvirus infections.

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