Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

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.

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

Journal of Virology ◽

10.1128/jvi.00733-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 4

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.

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Factors Influencing The Attenuation of Serotype 1 Marek's Disease Virus by Serial Cell Culture Passage and Evaluation of Attenuated Strains for Protection and Replication

Avian Diseases ◽

10.1637/8411-071908-reg.1 ◽

2009 ◽

Vol 53 (1) ◽

pp. 63-72 ◽

Cited By ~ 8

Author(s):

Ekaterina Dudnikova ◽

Anatoly Vlasov ◽

Svetlana Norkina ◽

Dmitry Kireev ◽

Richard L. Witter

Keyword(s):

Cell Culture ◽

Disease Virus ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Factors Influencing ◽

Culture Passage ◽

Serotype 1 ◽

Cell Culture Passage

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Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India

International Journal of Infectious Diseases ◽

10.1016/j.ijid.2007.06.008 ◽

2008 ◽

Vol 12 (2) ◽

pp. 190-197 ◽

Cited By ~ 38

Author(s):

A.V. Singh ◽

S.V. Singh ◽

G.K. Makharia ◽

P.K. Singh ◽

J.S. Sohal

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Mycobacterium Avium ◽

Human Population ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Healthy Human

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

Open Veterinary Journal ◽

10.4314/ovj.v10i2.4 ◽

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.

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Molecular characterization of Mycobacterium avium subsp. paratuberculosis (MAP) in dairy cattle of Nepal

Journal of Animal Science and Veterinary Medicine ◽

10.31248/jasvm2020.241 ◽

2020 ◽

Vol 5 (6) ◽

pp. 212-217

Author(s):

S. Singh ◽

◽

I. P. Dhakal ◽

U. M. Singh ◽

B. Devkota ◽

...

Keyword(s):

Dairy Cattle ◽

Molecular Characterization ◽

Mycobacterium Avium ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Restriction Endonuclease Analysis ◽

Cattle Population ◽

Control And Prevention ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Specific Sequences

Paratuberculosis caused by Mycobacterium avium subspecies paratuberculosis (MAP), is an economically important, endemic in many parts of the globe, and regarded as high prevalent disease of domestic and wild animals, especially ruminants, which is manifest as chronic granulomatous enteritis with decreased milk production, with serious cases resulting in progressive emaciation and death. Understanding the genetic variability of MAP, strains are important in diagnosis, epidemiological investigation, and therefore the formation of strategies for prevention and control of the disease. Thus, this study was designed to grasp the molecular characterization of MAP isolates of Nepal, as pioneer research of this area. Total of 46 MAP isolates obtained from cattle population of three different locations of dairy pocket areas of Chitwan, Nepal were typed using IS1311 polymerase chain reaction-restriction endonuclease analysis (PCR-REA) to research the MAP genotype of Nepal. The extracted DNA samples (n=46) were analyzed for the presence of MAP specific sequences (IS900) using PCR and DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. All the DNA samples were positive for the entire three MAP specific sequences based PCRs. This study revealed that ‘Bison type’ strain is the single most prevalent MAP genotype circulating within the domestic cattle population of Nepal. IS1311 PCR-REA showed that MAP DNA samples of Nepal origin belonged to ‘Bison type’, whereas, IS1311 L2 PCR-REA method showed similarity with "Indian Bison type" and different restriction profiles of ‘Bison type’ genotype as compared to non-Indian strains. The study concludes that in Nepal, "Bison type" MAP stains was prevalent in all the MAP samples obtained from dairy cattle. These results have important epidemiological implications regarding control and prevention of paratuberculosis in Nepal.

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