Efficacy of an inactivated influenza vaccine adjuvanted with Toll-like receptor ligands against transmission of H9N2 avian influenza virus in chickens.

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)- 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation.

The tumor microenvironment generated by Marek's disease virus suppresses interferon-gamma-producing gamma delta T cells.

The tumor microenvironment (TME) is generated by the cross-talk among tumor cells, immune system cells, and stromal cells. The TME generated by Marek's disease virus (MDV) is suggested to display an immunosuppressive milieu due to immune inhibitory molecules and cytokines which are possibly induced by MDV-transformed cells and regulatory T cells. Both anti-tumor and pro-tumor gamma delta (γδ) T cells are reported in human cancer. Although anti-tumor like and pro-tumor like γδ T cells are found in MDV-infected chickens at the later phase of infection, how the TME affects circulating and tissue-resident γδ T cells has not been investigated. Here, we demonstrated that the supernatant of the cultured splenocytes derived from MDV-challenegd chickens inhibited interferon (IFN)-γ production and CD25 expression by T cell receptor (TCR)γδ-stimulated tissue-resident γδ T cells, but the supernatant of the cultured MDV-transformed cell line did not affect γδ T cell activation. TCRγδ-stimulated circulating γδ T cells were influenced neither by the supernatant of the cultured splenocytes derived from MDV-challenegd chickens nor by the supernatant of the cultured MDV-transformed cell line. Taken together, activation and IFN-γ production by tissue-resident γδ T cells can be inhibited in the TME generated by MDV while tumor attracted circulating γδ T cells may not be influenced in activation and IFN-γ production by the TME generated by MDV.

CD4+TGFß+ cells infiltrated the bursa of Fabricius following IBDV infection, and correlated with a delayed viral clearance, but did not correlate with disease severity, or immunosuppression.

Introduction: Infectious Bursal Disease Virus (IBDV) causes immunosuppression in chickens. While B-cell destruction is the main cause of humoral immunosuppression, bursal T cells from IBDV-infected birds have been reported to inhibit the mitogenic response of splenocytes, indicating that some T cell subsets in the infected bursa have immunomodulatory activities. CD4+CD25+TGFß+ cells have been recently described in chickens that have immunoregulatory properties and play a role in the pathogenesis of Marek's Disease Virus. Methods: To evaluate if CD4+CD25+TGFß+ cells infiltrated the bursa of Fabricius (BF) following IBDV infection, and influenced the outcome of infection, birds were inoculated at either 2 days or 2 weeks of age with vaccine strain (228E), classic field strain (F52/70), or PBS (mock), and bursal cell populations were quantified by flow cytometry. Results: Both 228E and F52/70 led to atrophy of the BF, a significant reduction of Bu1+-B cells, and a significant increase in CD4+ and CD8α+ T cells in the BF, but only F52/70 caused suppression of immune responses to a test antigen in younger birds, and clinical signs in older birds. Virus was cleared from the BF more rapidly in younger birds than older birds. An infiltration of CD4+CD25+T cells into the BF, and elevated expression of bursal TGFß-1+ mRNA was observed at all time points following infection, irrespective of the strain or age of the birds, but CD4+TGFß+cells and CD4+CD25+TGFß+ cells only appeared in the BF at 28 dpi in younger birds. In older birds, CD4+TGFß+ cells and CD4+CD25+TGFß+ cells were present at earlier time points, from 7dpi following 228E infection, and from 14 and 28 dpi following F52/70 infection, respectively. Discussion: Our data suggest that an earlier infiltration of CD4+TGFß+ cells into the BF correlated with a delayed clearance of virus. However, the influx of CD4+TGFß+ cells and CD4+CD25+TGFß+ into the BF did not correlate with increased pathogenicity, or immunosuppression.

Inhibition of Marek's Disease Virus Replication and Spread by 25-hydroxycholesterol and 27-hydroxycholesterol In Vitro.

Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread.

Preventing bacterial disease in poultry in the post-antibiotic era: a case for innate immunity modulation as an alternative to antibiotic use.

The widespread use of antibiotics in the poultry industry has led to the emergence of antibiotic-resistant bacteria, which pose a significant health risk to humans and animals. These public health concerns, which have led to legislation limiting antibiotic use in animals, drive the need to find alternative strategies for controlling and treating bacterial infections. Modulation of the avian innate immune system using immunostimulatory compounds provides a promising solution to enhance poultry immune responses to a broad range of bacterial infections without the risk of generating antibiotic resistance. An array of immunomodulatory compounds have been investigated for their impact on poultry performance and immune responses. However, further research is required to identify compounds capable of controlling bacterial infections without detrimentally affecting bird performance. It is also crucial to determine the safety and effectiveness of these compounds in conjunction with poultry vaccines. This review provides an overview of the various immune modulators known to enhance innate immunity against avian bacterial pathogens in chickens, and describes the mechanisms involved.

Treatment with Toll-like Receptor (TLR) Ligands 3 and 21 Prevents Fecal Contact Transmission of Low Pathogenic H9N2 Avian Influenza Virus (AIV) in Chickens.

Transmission of H9N2 avian influenza virus (AIV) can occur in poultry by direct or indirect contact with infected individuals, aerosols, large droplets and fomites. The current study investigated the potential of H9N2 AIV transmission in chickens via a fecal route. Transmission was monitored by exposing naïve chickens to fecal material from H9N2 AIV-infected chickens (model A) and experimentally spiked feces (model B). The control chickens received H9N2 AIV. Results revealed that H9N2 AIV could persist in feces for up to 60-84 h post-exposure (PE). The H9N2 AIV titers in feces were higher at a basic to neutral pH. A higher virus shedding was observed in the exposed chickens of model B compared to model A. We further addressed the efficacy of Toll-like receptor (TLR) ligands to limit transmission in the fecal model. Administration of CpG ODN 2007 or poly(I:C) alone or in combination led to an overall decrease in the virus shedding, with enhanced expression of type I and II interferons (IFNs) and interferon-stimulating genes (ISGs) in different segments of the small intestine. Overall, the study highlighted that the H9N2 AIV can survive in feces and transmit to healthy naïve chickens. Moreover, TLR ligands could be applied to transmission studies to enhance antiviral immunity and reduce H9N2 AIV shedding.

Activated Chicken Gamma Delta T Cells Are Involved in Protective Immunity against Marek's Disease.

Gamma delta (γδ) T cells play a significant role in the prevention of viral infection and tumor surveillance in mammals. Although the involvement of γδ T cells in Marek's disease virus (MDV) infection has been suggested, their detailed contribution to immunity against MDV or the progression of Marek's disease (MD) remains unknown. In the current study, T cell receptor (TCR)γδ-activated peripheral blood mononuclear cells (PBMCs) were infused into recipient chickens and their effects were examined in the context of tumor formation by MDV and immunity against MDV. We demonstrated that the adoptive transfer of TCRγδ-activated PBMCs reduced virus replication in the lungs and tumor incidence in MDV-challenged chickens. Infusion of TCRγδ-activated PBMCs induced IFN-γ-producing γδ T cells at 10 days post-infection (dpi), and degranulation activity in circulating γδ T cell and CD8α+ γδ T cells at 10 and 21 dpi in MDV-challenged chickens. Additionally, the upregulation of IFN-γ and granzyme A gene expression at 10 dpi was significant in the spleen of the TCRγδ-activated PBMCs-infused and MDV-challenged group compared to the control group. Taken together, our results revealed that TCRγδ stimulation promotes the effector function of chicken γδ T cells, and these effector γδ T cells may be involved in protection against MD.

Determining the Protective Efficacy of Toll-Like Receptor Ligands to Minimize H9N2 Avian Influenza Virus Transmission in Chickens.

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1ß, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens.

Effect of treatment with Lactococcus lactis NZ9000 on intestinal microbiota and mucosal immune responses against Clostridium perfringens in broiler chickens.

Alterations in intestinal microbiota can modulate the developing avian intestinal immune system and, subsequently, may impact on resistance to enteric pathogens. The aim was to demonstrate that early life exposure to Lactococcus lactis, could affect either susceptibility or resistance of broilers to necrotic enteritis (NE). L. lactis NZ9000 (rL. lactis) pre-treatment at 1, 7, 14 and 21 days of age (DOA) led to a significant decrease in NE lesion scores in Clostridium perfringens infected chickens. C. perfringens Infection was associated with spatial and temporal decreases in mononuclear phagocytes and CD4+ αß T cells. However, rL. Lactis pre-treatment and subsequent C. perfringens infection led to a significant increase in mononuclear phagocytes, CD8α + γδ T, αß T cells (CD4+ and CD8α+) and B cells (IgM+, IgA+ and IgY+), as well as IL-12p40, IFN-γ and CD40. Differential expression of interleukin (IL)-6, IL-8, IL-10, IL-13, IL-18, IL-22, and transforming growth factor (TGF)-ß were observed in L. lactis treated chickens when compared to C. perfringens infected chickens. Microbiota analysis in C. perfringens infected chickens demonstrated an increase in abundance of Bacillota, Bacteroidota, Pseudomonadota and Actinomycetota. These findings suggests that modulation of the chicken intestinal immune system by L. lactis confers partial protection 30 against NE.

Corrigendum: Marek's disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response.

[This corrects the article DOI: 10.3389/fimmu.2022.973762.].

Marek's disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response.

The major histocompatibility complex (MHC) haplotype is one of the major determinants of genetic resistance and susceptibility of chickens to Marek's disease (MD) which is caused by an oncogenic herpesvirus; Marek's disease virus (MDV). To determine differential functional abilities of T cells associated with resistance and susceptibility to MD, we identified immunodominant CD4+TCRvß1 T cell epitopes within the pp38 antigen of MDV in B19 and B21 MHC haplotype chickens using an ex vivo ELISPOT assay for chicken IFN-gamma. These novel pp38 peptides were used to characterize differential functional abilities of T cells as associated with resistance and susceptibility to MD. The results demonstrated an upregulation of cytokines (IL-2, IL-4, IL-10) and lymphocyte lysis-related genes (perforin and granzyme B) in an antigen specific manner using RT-PCR. In the MD-resistant chickens (B21 MHC haplotype), antigen-specific and non-specific response was highly skewed towards Th2 response as defined by higher levels of IL-4 expression as well as lymphocyte lysis-related genes compared to that in the MD-susceptible chicken line (B19 MHC haplotype). Using CD107a degranulation assay, the results showed that MDV infection impairs cytotoxic function of T cells regardless of their genetic background. Taken together, the data demonstrate an association between type of T cell response to pp38 and resistance to the disease and will shed light on our understanding of immune response to this oncogenic herpesvirus and failure to induce sterile immunity.

Differential activation of chicken gamma delta T cells from different tissues by Toll-like receptor 3 or 21 ligands.

Gamma delta (γδ) T cells are highly enriched in mucosal barrier sites including intestinal tissues where microbial infections and tumors often originate in mammals. Human γδ T cells recognize stress antigens and microbial signals via their T cell receptor (TCR), natural killer (NK) receptors, and pattern recognition receptors. However, little is known about antigens or ligands capable of stimulating chicken γδ T cells. The results of the present study demonstrated that polyinosinic-polycytidylic acid (poly(I:C)), a Toll-like receptor (TLR)3 ligand, significantly induced upregulation of CD8α molecules on circulating and lung γδ T cells. Moreover, poly(I:C) stimulation induced interferon (IFN)-γ production from splenic and lung CD8α+ γδ T cells while Cytosine-phosphate-Guanine oligodeoxynucleotides (CpG-ODN) 2007, a TLR21 ligand, stimulation induced IFN-γ production by circulating γδ T cells. Neither poly(I:C) nor CpG-ODN 2007 stimulation elicited degranulation of γδ T cells. Additionally, the results revealed that CpG-ODN 2007 induced IFN-γ production from TCR-stimulated γδ T cells sorted from spleen. In our experiments, isopentenyl pyrophosphate (IPP), 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), or zoledronate (Zol) stimulation did not induce IFN-γ production or degranulation in γδ T cells. Taken together, a combination of CpG-ODN 2007 and anti-CD3ε monoclonal antibodies (mAbs) can stimulate chicken γδ T cells and induce production of IFN-γ by these cells while IFN-γ production by γδ T cells induced by stimulation of poly(I:C) needs signals from other cells. These results suggest that chicken γδ T cells can sense invading pathogens via TLRs and produce IFN-γ as a first line of defense.

Phenotypic characterization of gamma delta (γδ) T cells in chickens infected with or vaccinated against Marek's disease virus.

Marek's disease (MD) vaccines reduce the incidence of MD but cannot control virus shedding. To develop new vaccines, it is essential to elucidate mechanisms of immunity to Marek's disease virus (MDV) infection. In this regard, gamma delta (γδ) T cells may play a significant role in prevention of viral spread and tumor surveillance. Here we demonstrated that MDV vaccination induced interferon (IFN)-γ+CD8α+ γδ T cells and transforming growth factor (TGF)-ß+ γδ T cells in lungs. γδ T cells from MDV-infected chickens exhibited cytotoxic activity. Importantly, γδ T cells from the vaccinated/challenged group exhibited maximum cytotoxic activity following ex vivo stimulation. These results suggest that MDV vaccines activate effector γδ T cells which may be involved in the development of protective immune responses against MD. Further, it was demonstrated that MDV infection increases the frequency of a subpopulation of γδ T cells expressing membrane-bound TGF-ß in MDV-infected birds.

The severe acute respiratory syndrome coronavirus 2 non-structural proteins 1 and 15 proteins mediate antiviral immune evasion.

Infection with pathogenic viruses is often sensed by innate receptors such as Toll-Like Receptors (TLRs) which stimulate type I and III interferons (IFNs) responses, to generate an antiviral state within many cell types. To counteract these antiviral systems, many viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode non-structural proteins (NSPs) that mediate immune evasion. Using an overexpression system in A549 â€‹cells, we demonstrated a significant increase (p â€‹≤ â€‹0.0001) in Vesicular Stomatitis Virus (VSV)-EGFP reporter virus replication in cell lines overexpressing either the SARS-CoV-2 NSP1 or NSP15 when compared to control A549 â€‹cells. The increase in VSV-EGFP virus output was associated with a decrease in TLR2, TLR4 and TLR9 protein expression and a lack of antiviral protein production. Truncation of both NSP1 and NSP15 led to an increase in cellular TLR2, TLR4 and TLR9 as well as a decrease in TLR2 expression respectively. This observation can be attributed to the presence of a functional domain in NSP1 and NSP15 between amino acid (aa) 120-180 and aa 230-346, respectively. Both TLR3 and TLR9 ligands but not TLR2 ligand were highly effective at overcoming NSP1 and NSP15 functional interference based on significant decrease (p â€‹≤ â€‹0.0001) in VSV-EGFP virus replication. NSP1 or NSP15 intracellular interactions are likely low affinity interactions that can be easily disrupted by stimulating cells with specific TLR3 and TLR9 ligands. This report provides insights into the role of SARS-CoV-2 NSP1 and NSP15 in limiting specific TLR pathway activation, as an evasive mechanism against host innate responses.

Marek's Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2.

Marek's disease virus (MDV), an avian alphaherpesvirus, infects chickens, transforms CD4+ T cells, and induces immunosuppression early during infection. However, the exact mechanisms involved in MDV-induced immunosuppression are yet to be identified. Here, our results demonstrate that MDV infection in vitro and in vivo induces activation of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2). This exerts its inhibitory effects on T cell proliferation at day 21 post infection via PGE2 receptor 2 (EP2) and receptor 4 (EP4). Impairment of the MDV-induced T cell proliferation was associated with downregulation of IL-2 and transferrin uptake in a COX-2/PGE2 dependent manner in vitro. Interestingly, oral administration of a COX-2 inhibitor, meloxicam, during MDV infection inhibited COX-2 activation and rescued T cell proliferation at day 21 post infection. Taken together, our results reveal a novel mechanism that contributes to immunosuppression in the MDV-infected chickens.

Differential Virus-Specific IFN-Gamma Producing T Cell Responses to Marek's Disease Virus in Chickens With B19 and B21 MHC Haplotypes.

Marek's disease virus (MDV), the etiologic agent for Marek's disease (MD), causes a deadly lymphoproliferative disease in chickens. Causes of the well-documented association between genetically defined lines of chicken and resistance to MD remain unknown. Here, the frequencies of IFN-gamma producing pp38 and MEQ-specific T cell responses were determined in line N (B21 haplotype; MD-resistant) and line P2a (B19 haplotype, MD-susceptible) chickens after infection with vaccine and/or virulent (RB1B) strains of MDV using both standard ex vivo and cultured chIFN-gamma ELISPOT assays. Notably, MDV infection of naïve and vaccinated MD-resistant chickens induced higher frequencies of IFN-gamma producing MDV-specific T cell responses using the cultured and ex vivo ELISPOT assay, respectively. Remarkably, vaccination did not induce or boost MEQ-specific effector T cells in the susceptible chickens, while it boosted both pp38-and MEQ-specific response in resistant line. Taken together, our results revealed that there is a direct association between the magnitude of T cell responses to pp38 and MEQ of MDV antigens and resistance to the disease.

De Novo Cholesterol Biosynthesis and Its Trafficking in LAMP-1-Positive Vesicles Are Involved in Replication and Spread of Marek's Disease Virus.

Marek's disease virus (MDV) transforms CD4+ T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in chickens. While MDV-infected chickens have normal serum concentrations of cholesterol, their aortic tissues were found to have elevated concentrations of free and esterified cholesterol. Here, we demonstrate that infection of chicken embryonated fibroblasts (CEFs) with highly pathogenic MDV-RB1B increases the cellular cholesterol content and upregulates the genes involved in cholesterol synthesis and cellular cholesterol homeostasis using comprehensive two-dimensional gas chromatography-mass spectrometry and real-time PCR (RT-PCR), respectively. Using small pharmacological inhibitors and gene silencing, we established an association between MDV-RB1B replication and mevalonic acid, sterol, and cholesterol biosynthesis and trafficking/redistribution. We propose that MDV trafficking is mediated by lysosome-associated membrane protein 1 (LAMP-1)-positive vesicles based on short hairpin RNA (shRNA) gene silencing and the colocalization of LAMP-1, glycoprotein B (gB) of MDV, and cholesterol (filipin III) fluorescence signal intensity peaks. In conclusion, our results demonstrate that MDV hijacks cellular cholesterol biosynthesis and cholesterol trafficking to facilitate cell-to-cell spread in a LAMP-1-dependent mechanism.IMPORTANCE MDV disrupts lipid metabolism and causes atherosclerosis in MDV-infected chickens; however, the role of cholesterol metabolism in the replication and spread of MDV is unknown. MDV-infected cells do not produce infectious cell-free virus in vitro, raising the question about the mechanism involved in the cell-to-cell spread of MDV. In this report, we provide evidence that MDV replication depends on de novo cholesterol biosynthesis and uptake. Interruption of cholesterol trafficking within multivesicular bodies (MVBs) by chemical inhibitors or gene silencing reduced MDV titers and cell-to-cell spread. Finally, we demonstrated that MDV gB colocalizes with cholesterol and LAMP-1, suggesting that viral protein trafficking is mediated by LAMP-1-positive vesicles in association with cholesterol. These results provide new insights into the cholesterol dependence of MDV replication.

Dissecting the Mechanism of Intracellular Mycobacterium smegmatis Growth Inhibition by Platelet Activating Factor C-16.

Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C-16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis inside phagocytic cells of monocytic cell line, THP-1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signaling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signaling pathway, directly inhibited the growth of M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signaling pathway, increased survival of intracellular M. smegmatis. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogs, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner.

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

Viruses may hijack glycolysis, glutaminolysis, or fatty acid ß-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 ß-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 ß-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 ß-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.

Replication of Marek's Disease Virus Is Dependent on Synthesis of De Novo Fatty Acid and Prostaglandin E2.

Marek's disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates the fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEFs). In addition, MDV-infected cells contained high levels of fatty acids and showed increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titers by approximately 30-fold. Addition of the downstream metabolites, including malonyl-coenzyme A and palmitic acid, completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates the COX-2/prostaglandin E2 (PGE2) pathway, as evident by increased levels of arachidonic acid, COX-2 expression, and PGE2 synthesis. Inhibition of the COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using short hairpin RNA reduced MDV titers, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is a link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.IMPORTANCE Disturbances of the lipid metabolism in chickens infected with MDV contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates FAS and induces LD formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway, which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signaling pathways.