Truncation of gene F5L partially masks rescue of vaccinia virus strain MVA growth on mammalian cells by restricting plaque size

Modified vaccinia virus Ankara (MVA) is a candidate vaccine vector that is severely attenuated due to mutations acquired during several hundred rounds of serial passage in vitro. A previous study used marker rescue to produce a set of MVA recombinants with improved replication on mammalian cells. Here, we extended the characterization of these rescued MVA strains and identified vaccinia virus (VACV) gene F5L as a determinant of plaque morphology but not replication in vitro. F5 joins a growing group of VACV proteins that influence plaque formation more strongly than virus replication and which are disrupted in MVA. These defective genes in MVA confound the interpretation of marker rescue experiments designed to map mutations responsible for the attenuation of this important VACV strain.

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Biological and physico-chemical characterization of ultra high diluted Hydrastis canadensis and in vitro studies on mammalian cells

Allgemeine Homöopathische Zeitung ◽

10.1055/s-0037-1601241 ◽

2017 ◽

Vol 262 (02) ◽

pp. 2-76

Author(s):

N Chandrakar ◽

MK Temgire ◽

SG Kane ◽

AK Suresh ◽

J Bellare

Keyword(s):

Mammalian Cells ◽

Chemical Characterization ◽

In Vitro Studies ◽

Hydrastis Canadensis ◽

Physico Chemical

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Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

Infection and Immunity ◽

10.1128/iai.70.3.1121-1128.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1121-1128 ◽

Cited By ~ 32

Author(s):

Kent B. Marty ◽

Christopher L. Williams ◽

Linda J. Guynn ◽

Michael J. Benedik ◽

Steven R. Blanke

Keyword(s):

Cytotoxic Activity ◽

Serratia Marcescens ◽

Mammalian Cells ◽

Divalent Cations ◽

Recombinant Expression ◽

Cytotoxic Factor ◽

Culture Filtrates ◽

Type Strains

ABSTRACT Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.

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Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

Journal of Virology ◽

10.1128/jvi.01884-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2540-2552 ◽

Cited By ~ 52

Author(s):

Michael H. Lehmann ◽

Wolfgang Kastenmuller ◽

Judith D. Kandemir ◽

Florian Brandt ◽

Yasemin Suezer ◽

...

Keyword(s):

Vaccinia Virus ◽

Viral Vector ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Human Monocytic Cell Line ◽

Modified Vaccinia Virus Ankara ◽

Ccl2 Expression ◽

Human Monocytic Cell

ABSTRACT Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4+ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.

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B-1 cell: the precursor of a novel mononuclear phagocyte with immuno-regulatory properties

Anais da Academia Brasileira de Ciências ◽

10.1590/s0001-37652009000300013 ◽

2009 ◽

Vol 81 (3) ◽

pp. 489-496 ◽

Cited By ~ 11

Author(s):

José Daniel Lopes ◽

Mario Mariano

Keyword(s):

Mammalian Cells ◽

Giant Cells ◽

Mononuclear Phagocyte ◽

Different Types ◽

Series Of Experiments ◽

B1 Cells ◽

Regulatory Properties

Characterization of the origin, properties, functions and fate of cells is a fundamental task for the understanding of physiological and pathological phenomena. Despite the bulk of knowledge concerning the diverse characteristics of mammalian cells, some of them, such as B-1 cells, are still poorly understood. Here we report the results obtained in our laboratory on these cells in the last 10 years. After showing that B-1 cells could be cultured and amplified in vitro, a series of experiments were performed with these cells. They showed that B1 cells reside mostly in the peritoneal and pleural cavities, migrate to distant inflammatory foci, coalesce to form giant cells and participate in granuloma formation, both in vitro and in vivo. They are also able to present antigens to immunologically responsive cells and are endowed with regulatory properties. Further, we have also shown that these cells facilitate different types of infection as well as tumor growth and spreading. These data are presently reviewed pointing to a pivotal role that these cells may play in innate and acquired immunity.

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Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells

Vaccines ◽

10.3390/vaccines8010137 ◽

2020 ◽

Vol 8 (1) ◽

pp. 137 ◽

Cited By ~ 1

Author(s):

Vajini N. Atukorale ◽

Jerry P. Weir ◽

Clement A. Meseda

Keyword(s):

Vaccinia Virus ◽

Intergenic Region ◽

Vaccine Development ◽

Gc Content ◽

Serial Passage ◽

Sequencing Analysis ◽

Viral Glycoprotein ◽

Production Scale ◽

Modified Vaccinia Virus Ankara ◽

In Cells

The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) US6 gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the CP77 gene locus (MVA_009–MVA_013) and the I8R-G1L intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and CP77 sites. Sequencing analysis implicated US6 deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the I8R-G1L site, suggesting better accommodation of transgenes in this intergenic region. Thus, the I8R-G1L intergenic region may be more useful for transgene insertion for enhanced stability.

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Activation of platelet heparitinase by tumor cell-derived factors

Blood ◽

10.1182/blood.v78.3.789.789 ◽

1991 ◽

Vol 78 (3) ◽

pp. 789-796 ◽

Cited By ~ 10

Author(s):

A Haimovitz-Friedman ◽

DJ Falcone ◽

A Eldor ◽

V Schirrmacher ◽

I Vlodavsky ◽

...

Keyword(s):

Tumor Cells ◽

Mammalian Cells ◽

Human Platelets ◽

Native Form ◽

Heat Stable ◽

Inactive Form ◽

Cell Lining ◽

Mouse Lymphoma

Abstract The nature of the cooperation between platelets and tumor cells during the process of blood-borne metastasis is essentially unknown. In previous in vitro studies we showed that platelets participated in the formation of gaps in the endothelial cell lining, and that concomitantly heparan sulfate glycosaminoglycans were degraded by the platelet heparitinase, released on activation of platelets. In the current study we show that the ability to degrade proteoheparan sulfate derived from endothelial extracellular matrix is gradually eliminated when the number of human platelets is decreased from 5 x 10(7) to 10(6) cells/mL. When aliquots of conditioned media or lysates of either Eb or heat-inactivated ESb mouse lymphoma cells (both of which showed no heparanase activity) were added to freeze-thawed lysates of 10(6) platelets, a reappearance of platelet heparitinase activity was observed. A similar activation was not elicited by lysates of several normal mammalian cells. These data suggest that in its native form, a fraction of the platelet heparitinase is stored in an inactive form that can be activated by a factor secreted by lymphoma, but not by normal cells. Partial characterization of the heparitinase-activating factor showed that it is a heat-stable polyanionic molecule, devoid of proteolytic activity and resistant to both proteolytic and chondroitinase digestions. Activation of platelet heparitinase was also observed on coincubation with chondroitinases ABC and AC, suggesting that the inactive form of platelet heparitinase could result from a complex formation with a chondroitinase-sensitive proteoglycan. The lymphoma-derived heparitinase activating factor itself is, however, not a chondroitinase, because activity of chondroitinase could not be detected in Eb and ESb cells. A possible mechanism by which tumor cells recruit and regulate the activity of platelet heparitinase, and its relevance to the progression of blood borne metastasis, is discussed.

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Characterization of a Mutant HIV-1 Reverse Transcriptase Resistant to (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione (TIBO R82150)

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029400500410 ◽

1994 ◽

Vol 5 (4) ◽

pp. 278-281

Author(s):

H. Samanta ◽

R. Rose ◽

A. K. Patick ◽

C. M. Bechtold ◽

J. Trimble ◽

...

Keyword(s):

Reverse Transcriptase ◽

Nucleoside Reverse Transcriptase Inhibitor ◽

Serial Passage ◽

Transcriptase Inhibitor ◽

Viral Resistance ◽

Resistant Virus ◽

Reverse Transcriptase Inhibitor ◽

Hiv 1

A virus strain resistant to R82150, a non-nucleoside reverse transcriptase (NNRT) inhibitor (tetrahydro-imidazo [4,5, 1- jk] [1,4] benzodiazepine-2(1 H)-thione), was isolated following serial passage of HIV-1 RF in CEM-SS cells. The virus is cross-resistant to another non-nucleoside reverse transcriptase inhibitor, TGG-II-23A [1,4-dimethyl-1-[5,5-dimethyl-2-oxazoionyl]-naphthalen-2-one), but remains susceptible to AZT, DDI, D4T and phosphonoformate (PFA). DNA sequencing of reverse transcriptase genes from resistant virus indicated that R82150 selects for amino acid alterations Y181C and V108I. In vitro mutagenized reverse transcriptase and recombinant HIV-1 (pNL4-3) carrying either of the mutations have been generated. Genotypic and phenotypic analyses identified V108I as an unreported R82150-associated mutation. Both reverse transcriptase and viral resistance assays indicated that the resistance conferred by the V108I mutation is 7-fold less than that conferred by Y181C.

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Marker Rescue of the Host Range Restriction Defects of Modified Vaccinia Virus Ankara

Virology ◽

10.1006/viro.1998.9397 ◽

1998 ◽

Vol 251 (2) ◽

pp. 334-342 ◽

Cited By ~ 56

Author(s):

Linda S. Wyatt ◽

Miles W. Carroll ◽

Claus-Peter Czerny ◽

Michael Merchlinsky ◽

Jerry R. Sisler ◽

...

Keyword(s):

Vaccinia Virus ◽

Host Range ◽

Range Restriction ◽

Marker Rescue ◽

Modified Vaccinia Virus Ankara ◽

Host Range Restriction

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Development of Cell-Based Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A Protease Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.4.1381-1390.2005 ◽

2005 ◽

Vol 49 (4) ◽

pp. 1381-1390 ◽

Cited By ~ 9

Author(s):

Victoria Chung ◽

Anthony R. Carroll ◽

Norman M. Gray ◽

Nigel R. Parry ◽

Pia A. Thommes ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Vaccinia Virus ◽

Protease Inhibitors ◽

Expression System ◽

Recombinant Vaccinia Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Recombinant Vaccinia

ABSTRACT A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.

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