Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: OUTPUTS: Our project has 4 broad objectives that include (1) defining host/virus interactions important for equine infectious anemia virus (EIAV) replication; (2) use of EIAV as a model of lentiviral pathogenesis; (3) studying the biochemical basis of EIAV mutation; (4) examining EIAV evolution in vivo. Our work to define host/virus interactions has focused on the role of the EIAV S2 protein in disease expression. S2 is dispensable for replication in cell culture but is required for disease expression in the horse. Using yeast two hybrid and pull-down assays we demonstrated an interaction between EIAV S2 and the host cellular proteins OS9 and TBP-1. To continue investigating S2 function we switched to an analysis of cytokine/chemokine gene expression in equine macrophage infected with a highly virulent cloned virus versus an S2 deletion on that clone. Equine macrophages cultures were prepared using from 6 healthy donor horses. The macrophages were infected with EIAV at an MOI =3 on day 3 post-plating. RNA was prepared from cells at days 1 to 4 post-infection and the level of ten cytokines and chemokines were measured by quantitative PCR. The results of the study indicate that the S2 protein impacts of the expression levels of proinflammatory cytokines and chemokines. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Target audiences include virologists, infectious disease experts and experts in equine infectious diseases. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The conducted studies increased our understanding of the role of the equine infectious anemia virus S2 protein in disease expression. While we do not yet understand the mechanism of S2 action, it is clear that the S2 protein provides for enhanced expression of proinflammatory cytokines and chemokines during infection. Thus in the case of an EIAV infection, the virus may be subverting the proinflammatory response to enhance its replication. The proinflammatory response apparently does not play a completely protective role during EIAV infection.
Publications
- Covaleda, L., Fuller, F.J., Payne, S.L. 2009. EIAV S2 protein enhances pro-inflammatory cytokine and chemokine responses in infected macrophages. Virology. Accepted November 4, 2009 (VIRO-09-704R1).
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Progress 07/10/03 to 07/09/09
Outputs
OUTPUTS: Four broad objectives of this project include (1) defining host/virus interactions important for equine infectious anemia virus (EIAV) replication; (2) use of EIAV as a model of lentiviral pathogenesis; (3) studying the biochemical basis of EIAV mutation; (4) examining EIAV evolution in vivo. Over the past year we have focused our studies on EIAV pathogenesis by examining the roles of the EIAV S2 protein and the surface protein (SU) in disease expression. To investigate the possible impacts of S2 and SU on disease, we measured the levels of cytokine/chemokine gene expression in equine macrophage infected with highly virulent cloned virus and a variety of mutants. Equine macrophages cultures were prepared from healthy donor horses and were infected with EIAV on day 3 post-plating. For S2 studies RNA was prepared from cells at days 1 to 4 post-infection and the level of ten cytokines and chemokines were measured by quantitative PCR. For SU studies, RNA was prepared from 1 to 4 hours post-infection. The results of these assays suggest that highly virulent strains of EIAV impact the expression of cytokine and chemokines by at least 2 distinct routes. Binding of virulent (but not avirulent) virus strains significantly increases the expression of inflammatory cytokines in the target cells. At later times post-infection S2 stimulates expression of proinflammatory cytokines as well as chemokines. PARTICIPANTS: Lina Covaleda was a graduate student participation of NIH funded EIAV studies. In May, 2010 she completed her studies and graduated from Texas A&M University with a Ph.D. in Veterinary Microbiology. She continue to work on the project as a post-doctoral research associate. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. In the past year we have continued to develop and test quantitative real time PCR assays for a number of equine cytokines and chemokines. We are generating new models of virus-host cell interactions that contribute to EIAV pathogenesis.
Publications
- Covaleda, L, Gno B-T, Fuller, F.J. Payne, S. L. 2010. Identification of cellular proteins interacting with equine infectious anemia virus S2 protein. Virus Research. 151:235-239.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Our project has 4 broad objectives that include (1) defining host/virus interactions important for EIAV replication; (2) use of EIAV as a model of lentiviral pathogenesis; (3) studying the biochemical basis of EIAV mutation; (4) examining EIAV evolution in vivo. We are actively working on objectives 1 and 2 as there are central to a recently funded (9/06) NIH award. Our work to define host/virus interactions has focused on the role of the EIAV S2 protein in disease expression. S2 is dispensable for replication in cell culture but is required for disease expression in the horse. Using yeast two hybrid and pull-down assays we demonstrated an interaction between EIAV S2 and the host cellular proteins OS9 and TBP-1. To continue investigating S2 function we switched to an analysis of cytokine/chemokine gene expression in equine macrophage infected with our highly virulent EIAV 17 clone versus an S2 deletion on that clone. EMDM cultures were prepared from 6 different horses and were infected at an MOI =3 on day 3 post-plating. RNA was prepared from cells at days 1 to 4 post-infection and the level of ten cytokines and chemokines was measured by quantitative PCR. The results of the study indicate that the S2 protein does in fact have an impact of the expression levels of some proinflammatory cytokines and some chemokines. The levels of Interleukin (IL)-1 and , IL-6, IL-8, MIP-1, MCP-2 and IP-10 were significantly increased in EMDM infected with EIAV17b (p<0.05) compared with those infected with EIAV17bS2 at different time points. (IL)-1 and , were elevated significantly (p<0.05) only at day 2 post-infection. MIP-1 only at day 3 and 4. IL-6, IL-8 and MCP-2, were induced significantly at all time points and IP-10 at days 2 and 3. In contrast the levels of tumor necrosis factor- (TNF-), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1 (MIP-1) showed no statistically significant changes at any point after infection. We have recently begun using an equine whole genome oligonucleotide assay to examine changes in EMDM gene expression after infection with wild type and EIAV mutants. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. Our studies of EIAV S2 in equine macrophages are incomplete, but we have cloned and characterized the equine homologues of the human OS9,TBP-1 and NFkB2 genes. We have also developed and tested quantitative real time PCR assays for a number of equine cytokines and chemokines. We are currently working with a recently available equine whole genome oligonucleotide array to probe effects of EIAV infections. These assays should benefit other studies of equine infectious diseases.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
Our project has 4 broad objectives that include (1) defining host/virus interactions important for EIAV replication; (2) use of EIAV as a model of lentiviral pathogenesis; (3) studying the biochemical basis of EIAV mutation; (4) examining EIAV evolution in vivo. We are actively working on objectives 1 and 2 as there are central to a recently funded (9/06) NIH award. We continue to examine the role of EIAV S2 in disease expression via its interactions with the host cell proteins OS9, TBP-1 and NFkB. These cellular proteins are involved in various aspects of innate immunity and we hypothesize that the role of EIAV S2 is to modify the response of the macrophage to infection by favoring virus replication. During 2007, we examined S2 interactions with the equine homologues of OS9, TBP-1 and NFkB2. We showed that EIAV S2 interacts with equine OS9 and equine TBP-1 however we have not been able to detect an interaction between EIAV S2 and equine NFkB2. Also, we were not able
to repeat preliminary data, reported in last year's report, that S2 impacts the processing of equine NFkB2 in macrophages. To continue investigating S2 function in macrophages we are using a different investigative strategy that involves analyzing cytokine/chemokine gene expression in equine macrophage infected with our highly virulent EIAV 17 clone versus an S2 deletion on that clone. We have shown previously that both of these viruses replicate in cultured macrophages but that the S2-deletion mutation fails to replicate to high titers, or to cause clinical signs in our Shetland pony model of infection. Preliminary results indicate that there are indeed differences in cytokine expression, over a period of several days, post-infection between the two virus stocks.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. Our studies of EIAV S2 in equine macrophages are incomplete, but in the process of these studies we have cloned and characterized the equine homologues of the human OS9,TBP-1 and NFkB2 genes. We have also developed and tested quantitative real time PCR assays for a number of equine cytokines and chemokines. These assays should benefit other studies of equine infectious diseases.
Publications
- Allen, C.A., Payne, S.L., Harville, M., Cohen, N. and Russell, K.E. 2007. Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages. J. Immunol. Methods. 328:59-69.
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Progress 01/01/06 to 12/31/06
Outputs
Our project has 4 broad objectives that include (1) defining host/virus interactions important for EIAV replication; (2) use of EIAV as a model of lentiviral pathogenesis; (3) studying the biochemical basis of EIAV mutation; (4) examining EIAV evolution in vivo. We actively working on objectives 1 and 2 as there are central to a recently funded (9/06) NIH award. We are focusing on the role of EIAV S2 in disease expression via its interactions with the host cell proteins OS9, TBP-1 and NFkB. These cellular proteins are involved in various aspects of innate immunity and we hypothesize that the role of EIAV S2 is to modify the response of the macrophage to infection by favoring virus replication. During the past year we cloned the equine homologues to OS9, TBP-1 and NFkB. We have also identified reagents that are suitable for detecting the equine proteins in various assays. This was a major accomplishment, as it allows us to do our work in the most appropriate system,
the equine macrophage. We have also found a method suitable for transfecting equine macrophages. This is a major breakthrough. Last year I reported that a manuscript describing S2 interactions with OS9, TBP-1 and NFkB was in preparation but I decided to gather additional data addressing the biological function of S2 in hopes of reaching a higher impact journal. We have good preliminary data that S2 effects the processing of NFKB in macrophages and this novel information will be included in an upcoming publication. As regards our second objective, Use of the EIAV model to gain a better understanding of lentivirus pathogenesis. We did not tested any additional EIAV clones in vivo during 2006, due to a lack of funds. However we are currently are embarking on a major push in this area. Our in vitro data with the S2 protein will facilitate these studies in the coming year. As regards objective 4, an examination of EIAV evolution, we have continued to analyze data collected from rapid
passage experiments. In these experiments ponies were infected with avirulent virus and whole blood was passed to naive animals at 12 days post infection. A series of pony-to-pony passages was accomplished. We have completed the majority of sequence analysis (approximately 100 sequences per time point) and in 2006 we focused on collecting data for the TM glycoprotein. Earlier data pointed to mutations in one region of SU as playing a key role in disease development but the TM data is also revealing a role for particular TM sequences.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. In particular we believe that our studies of S2 point to a novel mechanism by which EIAV subverts the immune system. Such studies positively impact the development of therapeutics, diagnostics and vaccines.
Publications
- Fagerness, A.J., Flaherty, M.T., Perry, S.T., Jia, B., Payne, S.L., Fuller, F.J. 2006. The S2 accessory gene of equine infectious anemia virus is essential for expression of disease in ponies. Virology. 349:22-30.
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Progress 01/01/05 to 12/31/05
Outputs
Our EIAV research covers four broad objectives. The status of these studies are as follows: Objective 1. Define key host/virus interactions in EIAV replication. We have used a yeast 2 hybrid screen to identify three cellular proteins that interact with the EIAV S2 protein (a key virulence determinant). These proteins, OS9, TBP-1 and NFkB play roles in important cellular processes including innate immunity to viral infection. We have verified that EIAVS2 interacts with these proteins using Far Western Blots and have begun to map the interacting domains between S2 and the cellular targets. We are currently preparing a manuscript to report this data. We hypothesize that by interacting with these host cell proteins, S2 modulates the immune response in the infected animal, allowing for enhanced viral replication. We are currently trying to elucidate the overall biologic effects of S2 and hope to obtain funds to pursue these studies in vivo. Objective 2. Use the EIAV model
to gain a better understanding of lentivirus pathogenesis. We have tested a set of chimeric EIAV clones in our Shetland pony model. The chimeric viruses are genetic mixtures of a highly virulent EIAV strain and an avirulent EIAV strain. The studies clearly demonstrated that the surface glycoprotein (SU) is a major virulence determinant of EIAV. Our data also suggests that disease development cannot be predicted simply on the intrinsic ability of EIAV to replicate in equine macrophages. S2 and the viral long terminal repeats also play independent roles in disease development. This data was published in 2005. Objective 4. Examine EIAV evolution. We have analyzed data collected from rapid passage experiments. In these experiments ponies were infected with avirulent virus and whole blood was passed to naive animals at 12 days post infection. A series of pony to pony passages was accomplished. Blood samples were collected are various times and the virus populations existing in animals
without disease and in animals that did develop acute disease were analyzed by sequencing the envelope genes of approx. 100 viruses per time point. The data point to mutations in one region of SU as playing a key role in disease development. A manuscript describing these results is in preparation and we will apply for additional funding to examine the biologic effects of SU sequence variation.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. In particular we believe that our studies of S2 point to a novel mechanism by which EIAV subverts the immune system. Such studies positively impact the development of therapeutics, diagnostics and vaccines.
Publications
- Ball, J.M., Swaggerty, C.L., Pei, X-F., Lim, W-S., Xu, X., Cox, V.C., Payne, S.L. (2005). SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties. Virology. 331:132-144.
- Lim, W.-S., Payne, S. L., Edwards, J. F., Kim, I. and J. M. Ball. (2005). Differential Effects of Virulent and Avirulent Equine Infectious Anemia Virus on Macrophage Cytokine Expression. Virology. 332:295-306.
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Progress 01/01/04 to 12/31/04
Outputs
Our project proposed 4 broad objectives including: (1) define host/virus interactions important for EIAV replication; (2) use EIAV as a model of lentiviral pathogenesis; (3) study biochemical basis of EIAV mutation; (4) examine EIAV evolution in vivo. In the past year we have continued to make progress on objectives 1, 2 and 4. Objective 1: Using a yeast 2-hybrid screen we have identified 3 cellular proteins that interact with the EIAV S2-protein product. One of the strongly interacting proteins (OS9) is part of a genetic locus that is amplified in osteosarcoma, but whose cellular function is not clear. OS9 was identified twice in the 2-hybrid screen. We have confirmed the interaction between EIAV S2 and OS9 using a bacterial 2-hybrid screen and have produced mammalian and bacterial expression clones of OS9. We are in the process of performing in vitro protein interaction assays with S2 and OS9. Objective 2: We have completed studies and produced publications that
describe the role of EIAV SU protein in virulence and describe differences in the biologic activities of the SU proteins from virulent and avirulent EIAV clones. We have also begun to develop real time PCR assays for equine cytokines. We will use these assays to probe the cytokine responses of macrophages exposed to the EIAV SU protein. Objective 4: We have sequenced viral populations from two sets (7 total animals) of EIAV infected ponies. We can correlate changes in one small region of the SU protein to the development of disease.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. Such studies positively impact the development of therapeutics, diagnostics and vaccines.
Publications
- Payne, S.L., Pei, X.F., Jia, B., Fagerness, A., Fuller, F. (2004). Influence of Long terminal repeat and env on the virulence phenotype of equine infectious anemia virus. J. Virol. 78:2478-2485.
- Ball, J.M., Swaggerty, C.L., Pei, X-F., Lim, W-S., Xu, X., Cox, V.C., Payne, S.L. (2005). SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties. Virology. In Press.
- Lim, W.-S., Payne, S. L., Edwards, J. F., Kim, I. and J. M. Ball. Differential Effects of Virulent and Avirulent Equine Infectious Anemia Virus on Macrophage Cytokine Expression.(2005). Virology. In Press.
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Progress 01/01/03 to 12/31/03
Outputs
This project proposed four broad objectives concerning the retrovirus EIAV. In the past year we have pursued studies related to Objectives 1, 2 and 4. The status of these studies are as follows: Objective 1. Define key host/virus interactions in EIAV replication. We have performed the initial stages of a yeast two hybrid screen to find cellular proteins that interact with the EIAV S2 protein. Six yeast clones encoding containing presumptive interactors have been identified. Over the next two to three months these clones will be further characterized and tested. Objective 2. Use the EIAV model to gain a better understanding of lentivirus pathogenesis. This year we completed testing our first set of chimeric EIAV clones and have confirmed that major virulence determinants map to the envelope and long terminal repeat regions of the virus. The surface protein region was further delineated as a critical virulence factor. These results were prepared for publication.
Objective 4. Examine EIAV evolution in two systems. This year we completed the clinical phase of a rapid passage experiment. Samples from four animals have been collected and processed. Viral sequences are currently being obtained by polymerase chain reactions from these samples We have also obtained several hundred viral sequences from a previous rapid passage experiment.
Impacts
Studies of the retrovirus equine infectious anemia virus are providing detailed information regarding novel interactions between host and pathogen. Such studies positively impact the development of therapeutics, diagnostics and vaccines.
Publications
- Lim, W.-S., Edwards, J.F., Boyd, N.K. Payne, S.L., and Ball J.M. 2003. Simultaneous quantitation of equine cytokine mRNAs using a multi-probe ribonuclease protection assay. Veterinary Immunology and Immunopathology. 91:45-51.
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