Progress 07/01/08 to 06/30/13
Outputs
Target Audience: The target audience includes undergraduate and graduate students, members of the scientific community working in the area of viral immunology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? The results have been presented to the communities of interest through both oral and poster presentations worldwide. Results from the project have been published in manuscripts and book chapters. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have identified 47 bovine proteins, involved in immune function of professional antigen presenting cells (APC) that have been significantly altered after cytopathic (cp) bovine viral diarrhea virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II- related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that by modulating expression levels in multiple proteins related to immune responses BVDV could significantly compromise immune defense mechanisms resulting in uncontrolled immune activation or suppression. Using cp and ncp BVDV, we employed differential detergent fractionation and multidimensional protein identification technology (DDF-MudPIT) and identified the differentially expressed proteins in our dataset. We next used a high-throughput enrichment tool and canonical protein interaction pathways and networks to determine how the two biotypes affect and/or exploit phagocytosis and fluid phase uptake in bovine monocytes. Using a proteomic approach, we evaluated the effects of the BVDV biotypes on the expression of proteins involved in caveolae-mediated endocytosis, macropinocytosis and phagocytosis. In regard to the major cellular pathways affected by BVDV infection, despite some similarities in the protein expression patterns, significant strain-related differences that could be due to biological differences between cp and ncp BVDV have been detected. Our functional data by Flow Cytometry supports the effects of the two viral biotypes on the endocytic pathways proteins in bovine monocytes. Using proteomics, we evaluated the effect of cp and ncp BVDV infection of bovine monocytes to determine their role in viral immune suppression and uncontrolled inflammation. Proteins were isolated by differential detergent fractionation and identified by 2D-LC ESI MS/MS. We identified 137 and 228 significantly altered bovine proteins due to ncp and cp BVDV infection, respectively. Functional analysis of these proteins using the Gene Ontology (GO) showed multiple under- and over-represented GO functions in molecular function, biological process and cellular component between the two BVDV biotypes. Analysis of the top immunological pathways affected by BVDV infection revealed that pathways representing macropinocytosis signalling, virus entry via endocytic pathway, integrin signalling and primary immunodeficiency signalling were identified only in ncp BVDV- infected monocytes. In contrast, pathways like actin cytoskeleton signalling, RhoA signalling, clathrin-mediated endocytosis signalling and interferon signalling were identified only in cp BDVD- infected cells. Of the six common pathways involved in cp and ncp BVDV infection, acute phase response signalling was the most significant for both BVDV biotypes. Although, most shared altered host proteins between both BVDV biotypes showed the same type of change, integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3) were down- regulated by ncp BVDV and up- regulated by cp BVDV infection. Having undertaken a mass spectra-based approach, we identified significantly altered bovine proteins by comparing protein profiles before and after BVDV infection. Functional analysis of these proteins using the Gene Ontology (GO) showed multiple under- and over-represented GO functions in molecular function, biological process and cellular component categories between the two BVDV biotypes. Characterizing these proteins through pathway analysis showed that mitochondrial dysfunction and oxidative phosphorylation pathways were one of the top significant pathways affected by cp BVDV biotypes. We demonstrate that in contrast to ncp BVDV, cp BVDV biotype down-regulated the majority of proteins involved in those pathways. Our proteome-based findings have been extended and supported by flow cytometry and Western blotting approaches. The differential expression of pro- and anti-apoptotic proteins in BVDV-infected bovine monocytes can control the fate of infected cells and determine whether BVDV produce cytopathic effect or replicate non-cytopathically to establish persistent infection. Our recently published book chapter is a review of our attempts to implement the proteomic approach to study the expression of bovine proteins involved in the host immune responses to Bovine Viral Diarrhea Virus (BVDV) infection. This review discusses the various analysis methods used by our team that contributed to the identification of multiple critical proteins involved in the mechanisms of BVDV pathogenesis, professional antigen presentation and viral-host interactions. In addition, the review includes the main findings from our previous and current proteomics analyses. We evaluated the role of in vitro cytopathic and noncytopathic Bovine Viral Diarrhea Virus infection on the important function of professional antigen presenting cells, antigen uptake.To determine if the early phase of selective and non-selective antigen uptake in monocytes is regulated by PI3K-dependent signaling pathways, we evaluated the effect of PI3K inhibitor, LY294002 on the receptor-mediated antigen uptake and macropinocytosis in monocytes. In addition, the kinase protein expression levels have been assessed in bovine APC by western blotting analysis. Our data agree with previous observations that PI3K is constitutively expressed in innate immune cells. However, in contrast to the earlier reports that PI3K is rapidly activated by antigens, the expression levels of PI3K are decreased in the presence of FITC-OVA suggesting that FITC-OVA uptake, unlike various pathogens, does not involve the PI3K-dependent TLR signaling pathways. Instead, the decreased levels of PI3K in the presence of FITC-OVA correlated with significantly increased active FITC-OVA uptake in bovine monocytes supporting our hypothesis that PI3K is an endogenous regulator of antigen uptake in professional APC. The addition of LY increase expression levels of PI3K that correlated with insignificant macropinocytic activity in bovine monocytes. This finding indicates that PI3K is an endogenous suppressor of the signaling events involved in receptor-mediated uptake of FITC-OVA in bovine monocytes. BVDV entry significantly decreased FITC-OVA uptake while an established BVDV infection increased FITC-OVA uptake in monocytes, suggesting that BVDV use receptor-mediated endocytosis for its own cell entry. PI3K inhibition further increased FITC-OVA uptake only in CP BVDV-infected monocytes, suggesting that only CP BVDV biotype may utilize PI3K signaling in the establishment of productive infection in bovine monocytes. This finding correlates with previous findings that CP, but not NCP, induce INF signaling. Whether the exact stage of CP BVDV regulation of PI3K is during entry or immediately post-entry stage is subject to further investigation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Lee, S.-R.#, Nanduri, B.#, Pharr, G. T., Stokes, J. V. Pinchuk, L. M.* 2009. Bovine Viral Diarrhea Virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes. Biochim. Biophys. Acta. 1794:14-22
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Ammari, M. G., McCarthy, F. M., Nanduri, B., Pinchuk, L. M.* 2010. Analysis of Bovine Viral Diarrhea Virus-infected monocytes: identification of cytopathic and non-cytopathic biotype differences. BMC Bioinformatics, 11 (Suppl 6):1-13
- Type:
Book Chapters
Status:
Published
Year Published:
2012
Citation:
Mais Ammari, Fiona McCarthy, Bindu Nanduri, George Pinchuk and Lesya Pinchuk. 2012. Understanding the pathogenesis of cytopathic and noncytopathic Bovine Viral Diarrhea Virus infection using proteomics, Proteomic Applications in Biology, ISBN 978-953-307-613-3, INTECH, Open Access Publisher, pp.65-78.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2012
Citation:
Mais Ammari, Bovine viral diarrhea biotypes and their contribution to pathogenesis of the desease in susceptible cells , Dissertation submitted to the faculty of Mississippi State University, December 2012
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2011
Citation:
Ammari, M.*, Pinchuk, L. November, 2011. Apoptosis pathway in cytopathic and non-cytopathic Bovine Viral Diarrhea Virus infection. Fifth BVDV Symposium, San Diego, CA (oral presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Ammari, M.*, McCarthy, M., Nanduri, B., Pinchuk, G., Lee, S., Pharr, G., Pinchuk, L. 2009. Cytopathic and Non-Cytopathic Bovine Viral Diarrhea Viruses Differentially Modulate Proteins Involved in Professional Antigen Presentation in Bovine Monocytes, 1st International Conference on Immune Tolerance , Boston, MA, USA, October 25-27, 2009 (poster presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Ammari, M.*, McCarthy, F., Nanduri, B., Pinchuk, G., Pinchuk, L. 2010. Analysis of Bovine Viral Diarrhea Viruses-infected monocytes: identification of cytopathic and non-cytopathic differences. December 2010, CRWAD, Chicago (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Pinchuk, L*., Ammari, M., McCarthy, F., Nanduri, B. 2010. Proteomic analysis of Bovine Viral Diarrhea Virus-infected monocytes. December 2010, CRWAD, Chicago (poster presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2011
Citation:
Mais Ammari*, Bindu Nanduri, George Pinchuk, Lesya Pinchuk. 2011. Evaluation of mitochondrial proteins in apoptosis pathway in Bovine Viral Diarrhea Virus-infection: a proteomic approach, MCBIOS, The 8th Annual Conference of the MidSouth Computational Biology and Bioinformatocs Society, College Station, TX (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Ammari, M.*, McCarthy, F., Nanduri, B., Stokes, J, Pinchuk, L. 2010. Proteomic analysis of Bovine Viral Diarrhea Virus Infected Monocytes . Identification of Cytopathic and Non-Cytopathic Strain Differences, MCBIOS, The 7th Annual Conference of the MidSouth Computational Biology and Bioinformatocs Society, AR (poster presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Pinchuk, G., Stokes, J., Lee, S., Nanduri, B., Johnson, C., Shelton, M., Verkhratsky, A., Pinchuk, L. 2009. Proteomic analysis reveals altered expression of protein kinase and transport-related proteins in bovine monocytes infected with a cytopathic virus. Association of Southeastern Biologists Annual Meeting, Birmingham, AL (oral presentation).
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: To evaluate the role of in vitro cytopathic and noncytopathic Bovine Viral Diarrhea Virus infection on the proteins related to the professional antigen presentation and anti-viral defense. PARTICIPANTS: Individuals who worked on the project: Dr. Lesya Pinchuk: Principle investigator Mais Ammari, PhD student Collaborators and contacts: Dr. Bindu Nanduri: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. Fiona McCarthy: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. George Pinchuk:Collaborator in the Department of Sciences and Mathematics, Mississippi University for Women TARGET AUDIENCES: The target audience includes undergraduate and graduate students, members of the scientific community working in the area of viral immunology and bioinformatics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We evaluated the role of in vitro cytopathic and noncytopathic Bovine Viral Diarrhea Virus infection on the important function of professional antigen presenting cells, antigen uptake.To determine if the early phase of selective and non-selective antigen uptake in monocytes is regulated by PI3K-dependent signaling pathways, we evaluated the effect of PI3K inhibitor, LY294002 on the receptor-mediated antigen uptake and macropinocytosis in monocytes. In addition, the kinase protein expression levels have been assessed in bovine APC by western blotting analysis. Our data agree with previous observations that PI3K is constitutively expressed in innate immune cells. However, in contrast to the earlier reports that PI3K is rapidly activated by antigens, the expression levels of PI3K are decreased in the presence of FITC-OVA suggesting that FITC-OVA uptake, unlike various pathogens, does not involve the PI3K-dependent TLR signaling pathways. Instead, the decreased levels of PI3K in the presence of FITC-OVA correlated with significantly increased active FITC-OVA uptake in bovine monocytes supporting our hypothesis that PI3K is an endogenous regulator of antigen uptake in professional APC. The addition of LY increase expression levels of PI3K that correlated with insignificant macropinocytic activity in bovine monocytes. This finding indicates that PI3K is an endogenous suppressor of the signaling events involved in receptor-mediated uptake of FITC-OVA in bovine monocytes. BVDV entry significantly decreased FITC-OVA uptake while an established BVDV infection increased FITC-OVA uptake in monocytes, suggesting that BVDV use receptor-mediated endocytosis for its own cell entry. PI3K inhibition further increased FITC-OVA uptake only in CP BVDV-infected monocytes, suggesting that only CP BVDV biotype may utilize PI3K signaling in the establishment of productive infection in bovine monocytes. This finding correlates with previous findings that CP, but not NCP, induce INF signaling. Whether the exact stage of CP BVDV regulation of PI3K is during entry or immediately post-entry stage is subject to further investigation.
Publications
- Mais Ammari, Bovine viral diarrhea biotypes and their contribution to pathogenesis of the desease in susceptible cells, Dissertation submitted to the faculty of Mississippi State University, December 2012
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: To evaluate the role of in vitro cytopathic and noncytopathic Bovine Viral Diarrhea Virus infection on the proteins related to the professional antigen presentation and anti-viral defense. PARTICIPANTS: Individuals who worked on the project: Dr. Lesya Pinchuk: Principle investigator Mais Ammari, PhD student Collaborators and contacts: Dr. Bindu Nanduri: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. Fiona McCarthy: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. George Pinchuk:Collaborator in the Department of Sciences and Mathematics, Mississippi University for Women TARGET AUDIENCES: The target audience includes undergraduate and graduate students, members of the scientific community working in the area of viral immunology and bioinformatics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Having undertaken a mass spectra-based approach, we identified significantly altered bovine proteins by comparing protein profiles before and after BVDV infection. Functional analysis of these proteins using the Gene Ontology (GO) showed multiple under- and over-represented GO functions in molecular function, biological process and cellular component categories between the two BVDV biotypes. Characterizing these proteins through pathway analysis showed that mitochondrial dysfunction and oxidative phosphorylation pathways were one of the top significant pathways affected by cp BVDV biotypes. We demonstrate that in contrast to ncp BVDV, cp BVDV biotype down-regulated the majority of proteins involved in those pathways. Our proteome-based findings have been extended and supported by flow cytometry and Western blotting approaches. The differential expression of pro- and anti-apoptotic proteins in BVDV-infected bovine monocytes can control the fate of infected cells and determine whether BVDV produce cytopathic effect or replicate non-cytopathically to establish persistent infection. Our recently published book chapter is a review of our attempts to implement the proteomic approach to study the expression of bovine proteins involved in the host immune responses to Bovine Viral Diarrhea Virus (BVDV) infection. This review discusses the various analysis methods used by our team that contributed to the identification of multiple critical proteins involved in the mechanisms of BVDV pathogenesis, professional antigen presentation and viral-host interactions. In addition, the review includes the main findings from our previous and current proteomics analyses.
Publications
- Mais Ammari, Fiona McCarthy, Bindu Nanduri, George Pinchuk and Lesya Pinchuk. 2012. Understanding the pathogenesis of cytopathic and noncytopathic Bovine Viral Diarrhea Virus infection using proteomics, Proteomic Applications in Biology, ISBN 978-953-307-613-3, INTECH, Open Access Publisher, pp.65-78.
- Presentations: Ammari, M.*, Pinchuk, L. November, 2011. Apoptosis pathway in cytopathic and non-cytopathic Bovine Viral Diarrhea Virus infection. Fifth BVDV Symposium, San Diego, CA (oral presentation)
- Mais Ammari*, Bindu Nanduri, George Pinchuk, Lesya Pinchuk. 2011. Evaluation of mitochondrial proteins in apoptosis pathway in Bovine Viral Diarrhea Virus-infection: a proteomic approach, MCBIOS, The 8th Annual Conference of the MidSouth Computational Biology and Bioinformatocs Society, College Station, TX (oral presentation)
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: 1. To evaluate the role of in vitro CP and NCP BVDV infection in the phenotypic and functional maturation of monocyte-derived DC in dairy cattle. Phenotypic (MHC II, CD80/86, CD1, CD11c, MR, etc.) and functional (active endocytosis, capacity to directly stimulate B cells, and promote DC-dependent cytokine polarization in PBMC cultures) maturation will be examined. 2. To assess the DC maturation-related membrane, cytosolic and nuclear protein expression in infection with NCP and CP BVDV strains. PARTICIPANTS: Individuals who worked on the project: Dr. Lesya Pinchuk: Principle investigator Mais Ammari, PhD student Collaborators and contacts: Dr. Bindu Nanduri: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. Fiona McCarthy: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU TARGET AUDIENCES: The target audience includes undergraduate and graduate students, members of the scientific community working in the area of viral immunology and bioinformatics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using proteomics, we evaluated the effect of cp and ncp BVDV infection of bovine monocytes to determine their role in viral immune suppression and uncontrolled inflammation. Proteins were isolated by differential detergent fractionation and identified by 2D-LC ESI MS/MS. We identified 137 and 228 significantly altered bovine proteins due to ncp and cp BVDV infection, respectively. Functional analysis of these proteins using the Gene Ontology (GO) showed multiple under- and over-represented GO functions in molecular function, biological process and cellular component between the two BVDV biotypes. Analysis of the top immunological pathways affected by BVDV infection revealed that pathways representing macropinocytosis signalling, virus entry via endocytic pathway, integrin signalling and primary immunodeficiency signalling were identified only in ncp BVDV- infected monocytes. In contrast, pathways like actin cytoskeleton signalling, RhoA signalling, clathrin-mediated endocytosis signalling and interferon signalling were identified only in cp BDVD- infected cells. Of the six common pathways involved in cp and ncp BVDV infection, acute phase response signalling was the most significant for both BVDV biotypes. Although, most shared altered host proteins between both BVDV biotypes showed the same type of change, integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3) were down- regulated by ncp BVDV and up- regulated by cp BVDV infection.
Publications
- Ammari, M. G., McCarthy, F. M., Nanduri, B., Pinchuk, L. M.* 2010. Analysis of Bovine Viral Diarrhea Virus-infected monocytes: identification of cytopathic and non-cytopathic biotype differences. BMC Bioinformatics, 11 (Suppl 6):1-13
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: To evaluate the role of in vitro CP and NCP BVDV infection in the phenotypic and functional maturation of monocyte-derived DC in dairy cattle. Phenotypic (MHC II, CD80/86, CD1, CD11c, MR, etc.) and functional (active endocytosis, capacity to directly stimulate B cells, and promote DC-dependent cytokine polarization in PBMC cultures) maturation will be examined. To assess the DC maturation-related membrane, cytosolic and nuclear protein expression in infection with NCP and CP BVDV strains. PARTICIPANTS: Individuals who worked on the project: Dr. Lesya Pinchuk: Principle investigator John Stokes, Research Associate II Mais Ammari, PhD student Collaborators and contacts: Dr. Bindu Nanduri: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. Todd Pharr: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU TARGET AUDIENCES: The target audience includes undergraduate and graduate stuidents, members of the scientific community working in the area of viral immunology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using cp and ncp BVDV, we employed differential detergent fractionation and multidimensional protein identification technology (DDF-MudPIT) and identified the differentially expressed proteins in our dataset. We next used a high-throughput enrichment tool and canonical protein interaction pathways and networks to determine how the two biotypes affect and/or exploit phagocytosis and fluid phase uptake in bovine monocytes. Using a proteomic approach, we evaluated the effects of the BVDV biotypes on the expression of proteins involved in caveolae-mediated endocytosis, macropinocytosis and phagocytosis. In regard to the major cellular pathways affected by BVDV infection, despite some similarities in the protein expression patterns, significant strain-related differences that could be due to biological differences between cp and ncp BVDV have been detected. Our functional data by Flow Cytometry supports the effects of the two viral biotypes on the endocytic pathways proteins in bovine monocytes.
Publications
- Lee, S.-R.#, Nanduri, B.#, Pharr, G. T., Stokes, J. V. Pinchuk, L. M.* 2009. Bovine Viral Diarrhea Virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes. Biochim. Biophys. Acta. 1794:14-22
|
Progress 07/01/08 to 12/31/08
Outputs
OUTPUTS: 1. To evaluate the role of in vitro CP and NCP BVDV infection in the phenotypic and functional maturation of monocyte-derived DC in dairy cattle. Phenotypic (MHC II, CD80/86, CD1, CD11c, MR, etc.) and functional (active endocytosis, capacity to directly stimulate B cells, and promote DC-dependent cytokine polarization in PBMC cultures) maturation will be examined. 2. To assess the DC maturation-related membrane, cytosolic and nuclear protein expression in infection with NCP and CP BVDV strains. PARTICIPANTS: Individuals who worked on the project: Dr. Lesya Pinchuk: Principle investigator. John Stokes, Research Associate II Collaborators and contacts: Dr. Bindu Nanduri: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU Dr. Todd Pharr: Collaborator in the Department of Basic Sciences, College of Veterinary Medicine, MSU TARGET AUDIENCES: The target audience includes undergraduate and graduate stuidents, members of the scientific community working in the area of viral immunology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have identified 47 bovine proteins, involved in immune function of professional antigen presenting cells (APC) that have been significantly altered after cytopathic (cp) bovine viral diarrhea virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II- related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that by modulating expression levels in multiple proteins related to immune responses BVDV could significantly compromise immune defense mechanisms resulting in uncontrolled immune activation or suppression.
Publications
- Lee, S.-R.#, Nanduri, B.#, Pharr, G. T., Stokes, J. V. Pinchuk, L. M.* 2009. Bovine Viral Diarrhea Virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes. Biochim. Biophys. Acta. 1794:14-22
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