scholarly journals Attenuation of a virulent swine acute diarrhea syndrome coronavirus strain via cell culture passage

Virology ◽  
2019 ◽  
Vol 538 ◽  
pp. 61-70 ◽  
Author(s):  
Y. Sun ◽  
J. Cheng ◽  
Y. Luo ◽  
X.L. Yan ◽  
Z.X. Wu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Acute Diarrhea ◽  
Culture Passage ◽  
Cell Culture Passage ◽  
Diarrhea Syndrome

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 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 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.

scholarly journals Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage

2017 ◽  
Vol 201 ◽  
pp. 62-71 ◽  
Author(s):  
Chun-Ming Lin ◽  
Yixuan Hou ◽  
Douglas G. Marthaler ◽  
Xiang Gao ◽  
Xinsheng Liu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Strain ◽  
Porcine Epidemic Diarrhea Virus ◽  
Diarrhea Virus ◽  
Culture Passage ◽  
Porcine Epidemic Diarrhea ◽  
Cell Culture Passage

scholarly journals In vitro activities of azithromycin, clarithromycin, and other antibiotics against Chlamydia pneumoniae.

1996 ◽  
Vol 40 (11) ◽  
pp. 2669-2670 ◽  
Author(s):  
C C Kuo ◽  
L A Jackson ◽  
A Lee ◽  
J T Grayston
Keyword(s):  
Cell Culture ◽  
Chlamydia Pneumoniae ◽  
Complete Inhibition ◽  
Free Medium ◽  
Inclusion Formation ◽  
Culture Passage ◽  
Quinolone Antibiotics ◽  
Intracellular Concentrations ◽  
Cell Culture Passage

The in vitro susceptibilities of Chlamydia pneumoniae isolates to macrolide, tetracycline, and quinolone antibiotics were determined. Tetracycline, clarithromycin, and erythromycin had the lowest MICs in the first cell culture passage. Azithromycin required the lowest concentration for complete inhibition of inclusion formation on the second pass into antibiotic-free medium, likely reflecting its high intracellular concentrations.

Genome Analysis of Marek's Disease Virus Strain CVI-988: Effect of Cell Culture Passage on the Inverted Repeat Regions

1999 ◽  
Vol 43 (2) ◽  
pp. 182 ◽  
Author(s):  
B. J. L. Hooft van Iddekinge ◽  
L. Stenzler ◽  
K. A. Schat ◽  
H. Boerrigter ◽  
G. Koch
Keyword(s):  
Cell Culture ◽  
Disease Virus ◽  
Virus Strain ◽  
Genome Analysis ◽  
Inverted Repeat ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Culture Passage ◽  
Cell Culture Passage

scholarly journals An unusual plaque variant of rubella virus

1972 ◽  
Vol 70 (1) ◽  
pp. 49-53 ◽  
Author(s):  
Janine J. Gould ◽  
Gwenneth D. Laurence ◽  
M. Butler
Keyword(s):  
Cell Culture ◽  
Vaccine Strain ◽  
Rubella Virus ◽  
Kidney Cells ◽  
Plaque Size ◽  
Small Plaque ◽  
Infected Infant ◽  
Culture Passage ◽  
Cell Culture Passage ◽  
Virus Strains

SUMMARYIn a comparative study of 24 different rubella virus strains, all but three formed small plaques in RK 13 cell cultures; of these one was the vaccine strain HPV-77, one was isolated from a congenitally infected infant, and the third was recovered from an adult who contracted severe rubella while handling this congenital strain. Whereas the plaque size of the vaccine strain was stable after further passage in cell culture, the plaque size of the other two rapidly diminished when the virus was passed in monkey kidney cells, and one of them was also reduced by passage in RK 13 cells. Cell culture passage of the typical small plaque strains did not result in altered plaque size.

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