scholarly journals More cell culture passaged Camelpox virus sequences found resembling those of vaccinia virus

2020 ◽  
Vol 10 (2) ◽  
pp. 144-156
Author(s):  
Abdelmalik I. Khalafalla ◽  
Mohamed A. Al Hosani ◽  
Hassan Zackaria Ali Ishag ◽  
Salama S. Al Muhairi
Keyword(s):  
Cell Culture ◽  
Sequence Analysis ◽  
Vaccinia Virus ◽  
Field Isolate ◽  
Wild Type Virus ◽  
Wild Type ◽  
Body Protein ◽  
High Passage ◽  
Culture Passage ◽  
Cell Culture Passage

Background: Camelpox is the most infectious and economically important disease of camelids that causes significant morbidity and mortality rates. Several live attenuated vaccines against Camelpox virus (CMLV) are produced worldwide by passaging field isolates in cell culture. Sequence of a high passage Saudi isolate of CMLV was previously found closely resembled Vaccinia virus (VACV).Aim: To determine whether other high cell culture passage CMLV isolates are genetically resemble VACV and further to explore the possible mechanism of the resemblance.Methods: We performed polymerase chain reaction and DNA sequence analysis of A-type inclusion body protein (ATIP), L1R, and open reading frame (ORF) 185 genes on different cell culture passage levels of a field isolate, two high passage vaccines, wild-type, and reference strains of CMLV.Results: We demonstrate that additional two high passage attenuated vaccine candidate from Sudan and UAE likewise contain sequences  resembling VACV more than CMLV. Furthermore, sequence analysis of the ATIP gene of selected virus passages in cell culture revealed that the shift to VACV-like occurred between passage 11 and 20 and up to the 10th passage the genome still resembles wild-type virus. This observation was further confirmed by recombination analysis which indicated recombination events at ATIP and ORF185 genes occurred at higher passages.Conclusion: We confirmed that the cell culture passage CMLV turns to resemble VACV after cell culture passage and concluded that the  resemblance may not be a result of contamination or misidentification as previously thought but could be due to recombination events that occurred during the passage process. Keywords: Camelpox virus, cell culture passage, phylogenetic analysis.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vera Grotheer ◽  
Nadine Skrynecki ◽  
Lisa Oezel ◽  
Jan Grassmann ◽  
Joachim Windolf ◽  
...  
Keyword(s):  
Cell Culture ◽  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Cell Type ◽  
Differentiation Potential ◽  
Culture Passage ◽  
Cell Type Specific ◽  
Cell Culture Passage ◽  
Tissue Origin

AbstractThe need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-β signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-β signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-β signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

scholarly journals Reduced Susceptibility to VIRIP-Based HIV-1 Entry Inhibitors Has a High Genetic Barrier and Severe Fitness Costs

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Janis A. Müller ◽  
Anna Glöckle ◽  
Ali Gawanbacht ◽  
Matthias Geyer ◽  
Jan Münch ◽  
...  
Keyword(s):  
Cell Culture ◽  
Fusion Peptide ◽  
Viral Fitness ◽  
Genetic Barrier ◽  
Viral Susceptibility ◽  
Culture Passage ◽  
Cell Culture Passage ◽  
Replication Fitness ◽  
Hiv 1

ABSTRACTVIRIP has been identified as natural HIV-1 inhibitor targeting the gp41 fusion peptide. An optimized analogue (VIR-576) was effective in a phase I/II clinical trial and initial studies showed that HIV-1 resistance to VIRIP-based inhibitors has a high genetic barrier. Partially resistant CXCR4 (X4)-tropic HIV-1 NL4-3 variants could be obtained, however, after more than 15 months of passaging in MT-4 cells in the presence of another derivative (VIR-353). Sequence analyses identified the accumulation of seven mutations across the HIV-1 envelope glycoprotein but outside the gp41 fusion peptide. The authors suggested that the three initial alterations conferred resistance, while subsequent changes restored viral fitness. Here, we introduced these mutations individually and in combination into X4- and CCR5 (R5)-tropic HIV-1 constructs and determined their impact on VIR-353 and VIR-576 susceptibility, viral infectivity, replication fitness, and fusogenicity. We found that essentially all seven mutations contribute to reduced susceptibility to VIRIP-based inhibitors. HIV-1 constructs containing ≥4 changes were substantially more resistant to both VIRIP-based inhibitors and the VRC34.01 antibody targeting the fusion peptide. However, they were also much less infectious and fusogenic than those harboring only the three initial alterations. Furthermore, the additional changes attenuated rather than rescued HIV-1 replication in primary human cells. Thus, the genetic barrier to HIV-1 resistance against VIRIP-based inhibitors is higher than previously suggested, and mutations reducing viral susceptibility come at a severe fitness cost that was not rescued during long-term cell culture passage.IMPORTANCEMany viral pathogens are critically dependent on fusion peptides (FPs) that are inserted into the cellular membrane for infection. Initially, it was thought that FPs cannot be targeted for therapy because they are hardly accessible. However, an optimized derivative (VIR-576) of an endogenous fragment of α1-antitrypsin, named VIRIP, targeting the gp41 FP reduced viral loads in HIV-1-infected individuals. Characterization of HIV-1 variants selected during long-term cell-culture passage in the presence of a VIRIP derivative suggested that just three mutations in the HIV-1 Env protein might be sufficient for VIRIP resistance and that four subsequent changes restored viral fitness. Here, we show that all seven mutations contribute to reduced viral susceptibility to VIRIP-based inhibitors and demonstrate that the additional changes strongly impair rather than rescue HIV-1 infectivity, fusogenicity, and replication fitness. High genetic barrier to resistance and severe fitness cost support further clinical development of this class of antiviral agents.

scholarly journals Mutations in the Vaccinia Virus A33R and B5R Envelope Proteins That Enhance Release of Extracellular Virions and Eliminate Formation of Actin-Containing Microvilli without Preventing Tyrosine Phosphorylation of the A36R Protein

2003 ◽  
Vol 77 (22) ◽  
pp. 12266-12275 ◽  
Author(s):  
Ehud Katz ◽  
Brian M. Ward ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Envelope Protein ◽  
Actin Polymerization ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Virus Release ◽  
Type Virus ◽  
Wild Type ◽  
Extracellular Virus

ABSTRACT The spread of vaccinia virus in cell cultures is mediated by virions that adhere to the tips of specialized actin-containing microvilli and also by virions that are released into the medium. The use of a small plaque-forming A36R gene deletion mutant to select spontaneous second-site mutants exhibiting enhanced virus release was described previously. Two types of mutations were found: C-terminal truncations of the A33R envelope protein and a single amino acid substitution of the B5R envelope protein. In the present study, we transferred each type of mutation into a wild-type virus background in order to study their effects in vitro and in vivo. The two new mutants conserved the enhanced virus release properties of the original isolates; the A33R mutant produced considerably more extracellular virus than the B5R mutant. The extracellular virus particles contained the truncated A33R protein in one case and the mutated B5R protein in the other. Remarkably, both mutants failed to form actin tails and specialized microvilli, despite the presence of an intact A36R gene. The synthesis of the A36R protein as well as its physical association with the mutated or wild-type A33R protein was demonstrated. Moreover, the A36R protein was tyrosine phosphorylated, a step mediated by a membrane-associated Src kinase that regulates the nucleation of actin polymerization. The presence of large numbers of adherent virions on the cell surface argued against rapid dissociation as having a key role in preventing actin tail formation. Thus, the A33R and B5R proteins may be more directly involved in the formation or stabilization of actin tails than had been previously thought. When mice were inoculated intranasally, the A33R mutant was highly attenuated and the B5R mutant was mildly attenuated compared to wild-type virus. Enhanced virus release, therefore, did not compensate for the loss of actin tails and specialized microvilli.

Pre-clinical development of cell culture (Vero)-derived H5N1 pandemic vaccines

2008 ◽  
Vol 389 (5) ◽  
Author(s):  
M. Keith Howard ◽  
Otfried Kistner ◽  
P. Noel Barrett
Keyword(s):  
Cell Culture ◽  
High Yield ◽  
Wild Type Virus ◽  
Effective Vaccine ◽  
Type Virus ◽  
Wild Type ◽  
Yield Production ◽  
Homologous Virus ◽  
Clade 1 ◽  
Influenza H5n1

AbstractThe rapid spread of avian influenza (H5N1) and its transmission to humans has raised the possibility of an imminent pandemic and concerns over the ability of standard influenza vaccine production methods to supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains, produced at a variety of manufacturing scales, were demonstrated to be highly immunogenic in animal models without the need for adjuvant. The vaccines induce cross-neutralising antibodies and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from other clades. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high-yield production of a candidate pandemic vaccine.

scholarly journals Vaccinia Virus G1 Protein, a Predicted Metalloprotease, Is Essential for Morphogenesis of Infectious Virions but Not for Cleavage of Major Core Proteins

2004 ◽  
Vol 78 (13) ◽  
pp. 6855-6863 ◽  
Author(s):  
Camilo Ansarah-Sobrinho ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Protein A ◽  
Proteolytic Processing ◽  
Wild Type Virus ◽  
Sequence Information ◽  
Wild Type ◽  
Active Site Residues ◽  
Virus Particles ◽  
Core Proteins

ABSTRACT Genes encoding orthologs of the vaccinia virus G1 protein are present in all poxviruses for which sequence information is available, yet neither the role of the protein nor its requirement for virus replication is known. G1 was predicted to be involved in the cleavage of core proteins, based on a transfection study and the presence of an HXXEH motif found in a subset of metallopeptidases. In the present study, we engineered a recombinant vaccinia virus containing a single copy of the G1L gene with a C-terminal epitope tag that is stringently regulated by the Escherichia coli lac repressor. In the absence of inducer, expression of G1 was repressed and virus replication was inhibited. Rescue of infectious virus was achieved by expression of wild-type G1 in trans, but not when the putative protease active site residues histidine-41, glutamate-44, or histidine-45 were mutated. Nevertheless, the synthesis and proteolytic processing of major core and membrane proteins appeared unaffected under nonpermissive conditions, distinguishing the phenotype of the G1L mutant from one in which the gene encoding the I7 protease was repressed. Noninfectious virus particles, assembled in the absence of inducer, did not attain the oval shape or characteristic core structure of mature virions. The polypeptide composition of these particles, however, closely resembled that of wild-type virus. Full-length and shorter forms of the G1 protein were found in the core fraction of virus particles assembled in the presence of inducer, suggesting that G1 is processed by self-cleavage or by another protease.

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