An &lt;em&gt;Ex Vivo&lt;/em&gt; Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Dulwich, Katherine L; Asfor, Amin S.; Gray, Alice; Nair, Venugopal; Broadbent, Andrew

doi:10.3791/58489

Cited by 12 publications

(10 citation statements)

References 21 publications

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“…Moreover, activation of lymphocytes by soluble CD40L and anti-TCR-␣␤ antibody alters cell metabolism (30), which can potentially mask the effects of MDV infection on metabolism. Unactivated human or chicken lymphocytes undergo apoptosis in vitro, and only cell activation by various ligands can reduce cell death in lymphocyte culture models (31,32). Therefore, the effects of MDV infection on glycolysis and glutaminolysis were initially studied in CEFs, but these data should be confirmed in immune cells in future studies.…”

Section: Discussionmentioning

confidence: 99%

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus

Boodhoo

Kamble

Sharif

et al. 2020

J Virol

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Viruses may hijack glycolysis, glutaminolysis, or fatty acid β-oxidation of host cells to provide the energy and macromolecules required for efficient viral replication. Marek’s disease virus (MDV) causes a deadly lymphoproliferative disease in chickens and modulates metabolism of host cells. Metabolic analysis of MDV-infected chicken embryonic fibroblasts (CEFs) identified elevated levels of metabolites involved in glutamine catabolism, such as glutamic acid, alanine, glycine, pyrimidine, and creatine. In addition, our results demonstrate that glutamine uptake is elevated by MDV-infected cells in vitro. Although glutamine, but not glucose, deprivation significantly reduced cell viability in MDV-infected cells, both glutamine and glucose were required for virus replication and spread. In the presence of minimum glutamine requirements based on optimal cell viability, virus replication was partially rescued by the addition of the tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate, suggesting that exogenous glutamine is an essential carbon source for the TCA cycle to generate energy and macromolecules required for virus replication. Surprisingly, the inhibition of carnitine palmitoyltransferase 1a (CPT1a), which is elevated in MDV-infected cells, by chemical (etomoxir) or physiological (malonyl-CoA) inhibitors, did not reduce MDV replication, indicating that MDV replication does not require fatty acid β-oxidation. Taken together, our results demonstrate that MDV infection activates anaplerotic substrate from glucose to glutamine to provide energy and macromolecules required for MDV replication, and optimal MDV replication occurs when the cells do not depend on mitochondrial β-oxidation. IMPORTANCE Viruses can manipulate host cellular metabolism to provide energy and essential biosynthetic requirements for efficient replication. Marek’s disease virus (MDV), an avian alphaherpesvirus, causes a deadly lymphoma in chickens and hijacks host cell metabolism. This study provides evidence for the importance of glycolysis and glutaminolysis, but not fatty acid β-oxidation, as an essential energy source for the replication and spread of MDV. Moreover, it suggests that in MDV infection, as in many tumor cells, glutamine is used for generation of energetic and biosynthetic requirements of the MDV infection, while glucose is used biosynthetically.

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Section: Discussionmentioning

confidence: 99%

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus

Boodhoo

Kamble

Sharif

et al. 2020

J Virol

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“…Primary bursal cells were obtained from the BF of line 15 and line W birds as previously described [ 20 , 21 ]. Briefly, the BF was harvested aseptically post-mortem and washed in sterile PBS.…”

Section: Methodsmentioning

confidence: 99%

Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo

Asfor

Nazki

Reddy

et al. 2021

Viruses

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In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

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“…Primary bursal cells were obtained from the BF of line 15 and line W birds as previously described (20, 21). Briefly, the BF was harvested aseptically post-mortem and washed in sterile PBS.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were re-suspended in RPMI before undergoing centrifugation at 2,000 rpm for 20 min at 4°C over Histopaque 1077 (Sigma-Aldrich). Bursal cells that banded at the interface of the medium and histopaque were collected, washed in PBS, counted, and re-suspended in Iscove’ s modified Dulbecco’ s medium (IMDM) supplemented with FBS, chicken serum (Sigma-Aldrich), insulin transferrin selenium (Gibco), beta-mercaptoethanol (Gibco), penicillin, streptomycin, nystatin and chCD40L (20, 21) (complete B cell media).…”

Section: Methodsmentioning

confidence: 99%

Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammationin vivoand more rapid induction of pro-inflammatory responses in primary bursal cells stimulatedex vivo

Nazki

et al. 2021

Preprint

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In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p<0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

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An <em>Ex Vivo</em> Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Cited by 12 publications

References 21 publications

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus

Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo

Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammationin vivoand more rapid induction of pro-inflammatory responses in primary bursal cells stimulatedex vivo

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