Progress 02/15/23 to 02/14/24
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdoctoral fellows, farm managers and equine industry stakeholders Changes/Problems: As indicated in our previous progress reports, the proposed start date for this grant was February 15, 2019, but the funding was released on 11/05/2019 (NIFA [FR] Award Notification 2019-67016-29102; Reference 2020-00387). The project was further delayed due to the COVID-19 pandemic. During the COVID-19 pandemic, the LSU research facilities were either closed or allowed only limited access (i.e., restricted access to buildings and animal housing facilities). Subsequently, unexpected issues at the University of Illinois were identified on an IACUC inspection and required repair before the experiments could begin. Therefore, it was not possible to purchase stallions to begin the in vivo studies as planned before. Our collaborator at the University of Illinois has provided a tentative budget that surpasses the amount originally budgeted due to the inflationary effect. We are currently working on addressing this issue. We have considered the possibility of focusing only on the genetically susceptible group of stallions rather than on both (susceptible and resistant), as our aim is mainly focusing on establishing persistent infection in the former group. The postdoctoral scholar has also faced a significantly steep learning curve and a few technical issues that have delayed the progress of Aim 1. We had significant issues scheduling ourin vivostudies with our collaborator, Dr. Igor Canisso, Associate Professor of Theriogenology at the University of Illinois. We have patiently waited, but he informed us on December 2023 that he cannot perform the subcontract work in their facilities after several years of facility construction, IACUC, and IRB revisions, and requests that we can no longer pursue based on the timeline. We have consulted with another close collaborator of us, Dr. Reed Holyoak, Professor of Theriogenology at Oklahoma State University, but unfortunately, his facilities cannot hold the minimum number of animals required to generate relevant data. Thus, we have devised an alternative approach to complete the in vivo portion of the project (Objectives 2 and 3; deviation plan) by using the large, archived tissue bank we have generated on our previous USDA-NIFA project spanning 2013-2018 from experimental infections of stallions (n=13). Objectives 2 and 3 will be revised and will include novel approaches to study the role of CXCL16/CXCR6 and the immune microenvironment. Many of these were not available at the time this project was designed and submitted for funding consideration in 2017 and have become available in very recent years (2021 and beyond). These approaches will involve multiplex immunohistochemistry and in situ hybridization in combination with spatial transcriptomics tools that are state-of-the-art. This will also be a novel application in the field of animal health. Please see the attached detailed deviation plan. We have submitted a no-cost extension for one year. What opportunities for training and professional development has the project provided?We have hired Dr. Come Thieulent from France as a postdoctoral scholar with a start date of August 2, 2021. Dr. Thieulent was promoted from postdoctoral scholar to Assistant Research Professor in Diagnostic Innovation as of December 11th, 2023. How have the results been disseminated to communities of interest?Abstracts and presentations (7), and publications (1). What do you plan to do during the next reporting period to accomplish the goals?Aim 1: We will continue the experiments in progress. We will be testing the two models proposed in addition to performing single-cell RNA sequencing. We will complete this objective shortly and a manuscript is in preparation for publication. Aim 2: As indicated above we have abandoned the proposed in vivo studies and plan to use the archived formalin-fixed, paraffin-embedded, and snap-frozen tissues from our previous study. We have a large bank of well-characterized tissues from LTPI and short-term shedder stallions to be used to complete Aim 2 as originally planned. For this, we will combine multiplex IHC and ISH with quantitative pathology analysis using the Akoya PhenoImager HT 2.0 and inForm or Halo software. Additionally, in the absence of live CD8+ T lymphocytes, objectives 2-iii will be complemented with in vitro experiments designed to confirm the putative eca-mir-128 target (CXCL16) combined with analysis of eca-mir-128 at the tissue level via miRNAscope®. Aim 3: As indicated for Aim 2, we will use archived formalin-fixed, paraffin-embedded, and snap-frozen tissues from our previous study. We will implement the Visium spatial whole transcriptome platform to characterize transcriptomic signatures in the CD8+ T lymphocytes in situ/within tissue context, allowing us to assess not only their specific transcriptomic signatures but also relationships with other cell types at near single-cell resolution that may have a critical role during LTPI in the male reproductive tract. This deviation will allow us to address Aim 3.
Impacts
What was accomplished under these goals?
We have established a group of horses for the experiments delineated under Aim 1. These horses belong to the teaching herd at LSU. We have performed the genotypic and phenotypic analysis of peripheral blood mononuclear cells (PBMCs) to confirm animals from both susceptible and resistant groups. A total of 12 horses have been selected for furtherex-vivoandin-vitroexperiments, including three CXCL16S/S, three CXCL16S/r, and six CXCL16r/r (IACUC protocol #IACUCAM-23-091). We started to investigate our proposed "Model 1" under Aim 1. First,we purified CD3+ T cells from peripheral blood mononuclear cells (PBMCs) and determined that, while EAV can successfully infect CD3+ T cells in the entire PBMCs fraction in vitro, it is unable to infect purified CD3+ T cells. This observation indicates that an additional cell population from the PBMCs fraction (possibly CD14+ monocytes) is required for CD3+ T-cell susceptibility to EAV. Additionally, we are producing recombinant equine CXCL16 in order to evaluate if the presence of this cytokine can alter the permissiveness of purified CD3+ T cells to EAV in vitro. Purified CD3+ T cells have also been stimulated with four cytokines (IFNg, IFNb, TNFa, and IL-1b) obtained from a commercial company and phorbol 12-myristate 13-acetate (PMA) as a positive control. Gene expression of CXCL16 and CXCR6 will be measured by RT-qPCR. The expression of CXCR6 and CXCL16 in CD3+ T cells is also under investigation via flow cytometry using polyclonal antibodies produced by our laboratory and determine if stimulation with the aforementioned cytokines can upregulate CXCL16 and CXCR6 in CD3+ T cells. Moreover, we have demonstrated that pre-treatment of the PBMCs with an anti-CXCL16 polyclonal antibody blocks the infection of CD3+ T cells. Also, we have shown that pre-treatment with the chemical compound ML339, a selective CXCR6 antagonist, will prevent infection of CD3+ T cells by EAV. These experiments will be completed soon, complemented with point #3 below and the corresponding manuscript is in progress. To further characterize the phenotype of the susceptible CD3+ T lymphocytes under Aim 1, we will deviate from the original plan of utilizing flow cytometry, and we are now planning to perform single-cell RNA sequencing as this technology is nowadays readily available. This approach will circumvent some issues we have faced using antibodies to equine cellular markers to study the CXCL16/CXCR6 axis in EAV infection. We have assembled a team of individuals at the Genomics Core Facility, Pennington Biomedical Research Center (PBRC), and LSU to perform single-cell sequencing and analysis on infected PBMCs and sorted cells. This portion of the experiment is in progress. Our preliminary and published data has demonstrated that EAV primarily infects CD14+ monocytes in peripheral blood. We have demonstrated that CXCL16 serves as a receptor for EAV in equine monocytes. We are currently conducting experiments to determine if CD163, a macrophage-specific membrane scavenger receptor, serves as an additional cellular receptor for EAV. We have demonstrated that pre-treatment of PBMCs with PRRSV/CD163-IN-1, a chemical compound known toblock the interaction between PRRSV and porcine CD163, blocks EAV infection of equine CD14+ monocytes in vitro. Further experiments are in progress to confirm these results, including using an anti-CD163 antibody and the overexpression of equine CD163 in two cell lines that are resistant to EAV infection (DLD-1 and MCF7). Preventing the infection of CD14+ monocyte cells with the anti-CD163 antibody and/or making non-susceptible cells susceptible to EAV will confirm the role of CD163 in the infection of equine CD14+ monocytes. These studies are in progress.4. Finally, as described in the previous reporting period, we identified one additional receptor/accessory cellular receptor molecule required for EAV attachment and entryin vitro. This protein was identified as vimentin. We have performed the vimentin knockout in the E. Derm cells, which do not express CXCL16S. The absence of vimentin expression has been confirmed on the E. DermDVim cell line by immunofluorescence and is further confirmed by western immunoblotting. Time-course infection of the E. DermDVim and the E. Derm cell transfected with a control plasmid with EAV will be performed to confirm the role of Vimentin in EAV infectionin vitro. We have purchased an Akoya PhenoImager HT 2.0 for the Histology/IHC section at the Louisiana Animal Disease Diagnostic Laboratory. This instrument will be used in Aims 2 and 3 with the modifications described. This instrument will allow whole slide scanning and quantitative analysis of tissue sections using multiplex IHC and ISH as proposed in the study. This equipment offers unparalleled multispectral imaging and unmixing of fluorescence signals compared to other systems, with the capability of spectrally unmixing up to 9 colors. This is the key to accurate biomarker quantitation to compensate for optical spectral bleed-through among channels and isolate signals from background autofluorescence. Its integration with inForm® analysis software and other quantitative pathology software packages with trainable machine-learning algorithms for unbiased, computer-based quantitative analysis of features of interest, will allow us to quantitate phenotypic markers and perform spatial analysis of cell populations as proposed. We are planning to purchase a Nikon Ti inverted fluorescence microscope with fluorescence capabilities of up to 4 colors. This equipment is needed for partial fulfillment of Aim 1 (cell-based assays using transfection experiments) as well as rapid evaluation of markers to be optimized for Aims 2 and 3. We have proposed a plan deviation for Aims 2 and 3 and requested a one-year no-cost extension for this grant.
Publications
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Progress 02/15/22 to 02/14/23
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdoctoral fellows, farm managers and equine industry stakeholders Changes/Problems: As indicated in our previous progress reports, the proposed start date for this grant was February 15, 2019, but the funding was released on 11/05/2019 (NIFA [FR] Award Notification 2019-67016-29102; Reference 2020-00387). The project was further delayed due to the COVID-19 pandemic. During the COVID-19 pandemic, the LSU research facilities were either closed or allowed only limited access (i.e., restricted access to buildings and animal housing facilities). Subsequently, unexpected issues at the University of Illinois were identified on an IACUC inspection and required repair before the experiments could begin. Therefore, it was not possible to purchase stallions to begin the in vivo studies as planned before. Our collaborator at the University of Illinois has provided a tentative budget that surpasses the amount originally budgeted due to the inflationary effect. We are currently working on addressing this issue. We have considered the possibility of focusing only on the genetically susceptible group of stallions rather than on both (susceptible and resistant), as our aim is mainly focusing on establishing persistent infection in the former group. The postdoctoral scholar has also faced a significantly steep learning curve and a few technical issues that have delayed the progress of Aim 1. In summary, the COVID-19 pandemic has pushed us back at least 12 months. Thus, we have not utilized the budget as projected. We hope to get back on track with the project this year. What opportunities for training and professional development has the project provided?1. We have hired Dr. Come Thieulent from France as a postdoctoral scholar with a start date of August 2, 2021. Dr. Thieulent has remained the postdoctoral scholar for this project to date. How have the results been disseminated to communities of interest?Abstracts, presentations, and publications. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on all three objectives now that the COVID-19 pandemic is under control, the University of Illinois seems to have renovated the facilities, and will do our best to compensate for the time lost. Aim 1: We will continue the experiments in progress. We will be testing the two models proposed in addition to performing single-cell RNA sequencing. Aim 2: We anticipate initiating the in vivo studies described in the proposal by the end of this spring, which will allow us to euthanize the first group of stallions before the end of 2023. Aim 3: We expect to perform experiments related to this aim corresponding to the first group of stallions to be analyzed following experimental infection.
Impacts
What was accomplished under these goals?
We have been working on Aim 1 and identified one additional receptor/accessory cellular receptor molecule required for EAV attachment and entry, as described in the previous reporting period. This protein appears to be vimentin. We have performed further work to characterize this protein. We are developing a biotin-labeled EAV in our laboratory to assess its binding to vimentin. Two protocols have been tested so far, DSPE-PEG(2000)-biotin and EZ-Link sulfo-NHS-biotin. However, both failed to label the biotin to EAV. An optimized protocol for EZ-Link sulfo-NHS-biotin will be conducted in order to label EAV with biotin. We have performed Western blot, plaque assays, and flow cytometry on multiple different cell lines infected with EAV and with variable levels of vimentin expression. We demonstrated that the virus replicates at low levels, and its replication is abrogated in cells expressing low levels (HEK-293T) or no vimentin (DLD-1 and MCF-7). These results confirmed that vimentin might play a primordial role in EAV infection. Equine dermis (E. Derm) cells were sent to collaborators at Kansas State University to knock out the equine vimentin gene. The knockout process is in progress and will take 3 to 4 months to generate a knockout cell line. Once this knockout cell line is obtained, additional experiments will be performed to assess the role of vimentin in EAV entry. Finally, we evaluated the effect of pre-treating equine endothelial cells with increasing concentrations of a monoclonal anti-vimentin antibody. While a reduction of viral titers was observed by plaque assay, we were not able to confirm whether this treatment blocked EAV entry by Western blot. This is likely due to the low sensitivity of the method, and additional incubation times (30 minutes to 2 hours) will be performed with an increase in protein concentration. We will also perform this experiment with a polyclonal anti-vimentin antibody.Despite the problem encountered, alternatives were found, and we expect to finalize this project at the end of 2023. In relation to Aim 1, we have established a group of horses for these experiments from the teaching herd at LSU. We have performed genotypification and phenotypic analysis of peripheral blood mononuclear cells to confirm animals from both susceptible and resistant groups. The postdoctoral scholar has re-established the flow cytometry and sorting techniques needed for this aim. To determine the role of CXCL16/CXCR6 in Aim 1, we proposed stimulating peripheral blood mononuclear cells with various cytokines. To address this, we have obtained various equine cytokines available commercially and which have previously been demonstrated to upregulate the expression of CXCL16 or CXCR6 based on the existing literature (human and mice). Eight cytokines (IL-12, IL-1b, IL-8, IL-4, IL-13, TNFa, IFNb, and INFg) will be tested at different concentrations, and the expression level of CXCL16 and CXCR6 will be measured by flow cytometry and gene expression quantification by RT-qPCR. This experiment will allow us to determine if the CD3+ cells can express CXCL16 and CXCR6 under various stimulations. All the cytokines were received, and we are currently finalizing the protocol of peripheral blood mononuclear cell stimulation. To further characterize the phenotype of the susceptible CD3+ T lymphocytes under Aim 1, we will deviate from the original plan of utilizing flow cytometry, and we are considering performing this work using single-cell RNA sequencing as this technology is nowadays readily available and will circumvent issues we have faced with antibodies to equine cellular markers. We have assembled a team of individuals at the Genomics Core Facility, Pennington Biomedical Research Center (PBRC), LSU to perform the sequencing and analysis on infected and sorted cells. This portion of the experiment is in progress. Dr. Carossino (anatomic pathologist) has started to implement multiplex in situ hybridization protocols in the Louisiana Animal Disease Diagnostic Laboratory, which will be used for Aims 2 and 3 of this project. We have submitted one abstract to the AAVLD Annual Meeting 2022, one to the CRWAD 2023, and one to an LSU-hosted event on International Education in 2022. These were all oral presentations delivered by the postdoctoral scholar. We are preparing an abstract submission to the 2023 NIDO meeting to take place in Switzerland in 2023. We are working with our collaborator (Dr. Igor Canisso) at the University of Illinois on acquiring stallions for the in vivo studies described in Aims 2 and 3. There were some delays in this regard as the University of Illinois would not be able to provide a budget for Dr. Canisso until the renovation of their facilities to hold the stallions was completed. Recent communication with Dr. Canisso indicates that renovations are nearly completed and that the experimental work can begin in May 2023.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Thieulent CJ, Carossino M, Balasuriya UBR, Graves K, Bailey E, Eberth J, Canisso IF, Andrews FM, Keowen ML, Go YY. Development of a TaqMan� Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions. Front Genet. 2022 Apr 13;13:871875. doi: 10.3389/fgene.2022.871875. PMID: 35495124; PMCID: PMC9043104.
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
1. Fang, Y., and Balasuriya, U. B. R. 2022. Arteriviruses (Chapter 29). In Fields Virology (Seventh Edition), Edited by David M. Knipe and Peter M. Howley. Wolters Kluwer / Lippincott Williams & Wilkins, Philadelphia, PA.
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
2. U. B. R. Balasuriya, Y. Y. Go and M. Carossino. Chapter 61. Coronaviridae and Tobaniviridae In: Veterinary Microbiology, 4th edition, S. McVey, M. Kennedy, M.M. Chengappa and R. Wilkes (eds), Wiley-Blackwell, October 11, 2022
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
3. U. B. R. Balasuriya, M. Carossino and Y. Y. Go. Chapter 62. Arteriviridae and Roniviridae. In: Veterinary Microbiology, 4th edition, S. McVey, M. Kennedy, M.M. Chengappa and R. Wilkes (eds), Wiley-Blackwell, October 11, 2022
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Progress 02/15/21 to 02/14/22
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdoctoral fellows, farm managers and equine industry stakeholders Changes/Problems: As indicated in our previous progress reports, the proposed start date for this grant was February 15, 2019, but the funding was released on 11/05/2019 (NIFA [FR] Award Notification 2019-67016-29102; Reference 2020-00387). The project was further delayed due to the COVID-19 pandemic. During the COVID-19 pandemic, the LSU research facilities were either closed or allowed only limited access (i.e., restricted access to buildings and animal housing facilities). Therefore, it was not possible to purchase stallions to begin the in vivo studies as planned before, and there were also significant delays to hire personnel and perform in vitro experiments in the laboratory. The graduate student from Sri Lanka who was supposed to work on this project last Fall was not granted the visa to enter the USA. Instead of him, we were able to hire a new postdoctoral scholar from France. In summary, the COVID-19 pandemic has pushed us back at least 12 months. Thus, we have not utilized the budget as projected. We hope to get back on track with the project this year. What opportunities for training and professional development has the project provided?1. Dr. Mariano Carossino became a tenure track faculty (Assistant Professor) in the Department of Pathobiological Sciences (PBS), School of Veterinary Medicine, and Louisiana Animal Disease Diagnostic Laboratory with a start date of April 5, 2021. He will continue to be the Co-PI and work on the project. 2. have hired Dr. Come Thieulent from France as a postdoctoral scholar with a start date of August 2, 2021. How have the results been disseminated to communities of interest?Abstracts, presentations, and publications. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on all three objectives now that the COVID-19 pandemic is under control. Aim 1: We will embark on the experimentation related to this aim and test our two model systems proposed. Aim 2: We will initiate the in vivo studies described in the proposal this spring. The first group of stallions will be euthanized by the end of 2022. Aim 3: We expect to perform experiments related to this aim corresponding to the first group of stallions to be analyzed following experimental infection.
Impacts
What was accomplished under these goals?
We have been working on Aim 1 and identified one additional receptor/accessory cellular receptor molecule required for EAV attachment and entry. This protein appears to be vimentin, and the role of this protein in EAV attachment and entry has been characterized. A manuscript detailing this work is in preparation. In relation with Aim 1, we have been working in establishing a group of horses for these experiments from the teaching herd at LSU. We started using some of these animals to train the new postdoctoral scholar (see below) and re-establish flow cytometry and sorting techniques needed for this aim. We have used the teaching herd as well as archived horse DNA to establish and validate a genotyping real-time PCR assay for genotypification of stallions based on the CXCL16 genotype. This assay is important for 2 main reasons: 1) for breeders and other stakeholders, since it allows to assess those stallions at highest risk of becoming persistently infected following exposure and, consequently, inform on targeted vaccination practices against EAV; and 2) use as a rapid tool to select horses for experimental infection under Aims 2 and 3. A manuscript describing this work has been generated and in press (see below). Dr. Carossino (anatomic pathologist) has started to implement multiplex immunohistochemistry protocols in the Louisiana Animal Disease Diagnostic Laboratory, which will be used for Aims 2 and 3 of this project. We have submitted one abstract to the Nidovirus 2021 meeting (hold virtually); poster was presented virtually. We have submitted one abstract to CRWAD 2021, and data was presented via Zoom. We had a project meeting with the USDA-NIFA project leaders on December 20, 2021, and provided an update on the project. We are working with our collaborator (Dr. Igor Canisso) at the University of Illinois on acquiring stallions for the in vivo studies described in Aims 2 and 3. The budget will be revised and reallocated to Dr. Canisso.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Thieulent, C. J., Carossino, M, Balasuriya, U. B. R., Graves, K., Bailey, E., Eberth, J., Canisso, I. F., Andrews,F. M., Keowen, M., and Go, Y. Y. 2022. Development of a TaqMan� allelic discrimination PCR assay for rapid detection of equine CXCL16 allelic variants associated with the establishment of long-term equine arteritis virus carrier state in stallions. Frontiers in Genetics (Livestock Genomics Section; In Press).
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Progress 02/15/20 to 02/14/21
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdoctoral fellows, farm managers and equine industry stakeholders. Changes/Problems: As indicated in our previous progress report, the proposed start date for this grant was February 15, 2019, but the funding was released on 11/05/2019 (NIFA [FR] Award Notification 2019-67016-29102; Reference 2020-00387). The project was further delayed due to the COVID-19 pandemic. During the COVID-19 pandemic, the LSU research facilities were either closed or allowed only limited access (i.e., restricted access to buildings and animal housing facilities). Therefore, it was not possible to purchase stallions to begin the in vivo studies as planned before. The graduate student from Sri Lanka could not start his program last Fall. He is expected to join the research program this Fall. The COVID-19 pandemic delayed advertising for a new postdoctoral scholar to be hired. XV International Nidovirus Symposium (Nido2020) was postponed from May 10-14, 2020, to June 7-8, 2021 (Nido2021). This meeting will now be held virtually (via Zoom). Two original abstracts that were submitted in 2020 will now be presented at this virtual meeting. In summary, the COVID-19 pandemic has pushed us back at least 9-10 months. Thus, we have not utilized the budget as projected. We hope to get back on track with the project this year. What opportunities for training and professional development has the project provided? Dr. Mariano Carossino is a postdoctoral scholar and anatomic pathology resident at the LADDL. He was interviewed for a tenure track faculty position (Assistant Professor) in the Department of Pathobiological Sciences (PBS), School of Veterinary Medicine. He will be starting his faculty job in April 2021 and will continue to be the Co-PI and work on the project. One graduate student (Dr. Devinda Wickramasingha from Sri Lanka) was admitted to our departmental (PBS) graduate program to work on this project. He was supposed to start the program in Fall 2020. However, due to the COVID-19 pandemic, he could not obtain his visa on time to enroll in classes. He is planning to join the lab in Fall 2021. We are in the process of hiring a new postdoc to work on this project within the next three months. The position has already been advertised. Ms. Lillian Miller (veterinary student) has been working on the project and she helped develop the miRNAscope assay to detect MicroRNA eca-mir-128 in the stallion reproductive tract of the stallions. How have the results been disseminated to communities of interest?Abstracts and presentations. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on all three objectives as soon as the COVID-19 outbreak is under control. We will initiate the in vivo studies described in the proposal.
Impacts
What was accomplished under these goals?
We purchased an advanced immunohistochemical (IHC) staining instrument (Leica BondRXm) for this project in August 2020. The instrument is installed and validated by Leica. Protocols for performing automated in situ hybridizations (singleplex and duplex) have been developed and tested. These protocols will be extensively used in the proposed project. We have optimized all necessary IHC protocols needed for objectives 2 and 3 in the laboratory using the new Leica BondRXm. We have used tissues from three EAV persistently infected stallions and one uninfected stallion that we collected last year to optimize IHC protocols on the new instrument. We have established the novel miRNAscope® assay to detect MicroRNA eca-mir-128 in the stallion reproductive tract. Our laboratory was one of the few involved in the validation of this novel technique. Archived tissues from 17 stallions have been tested for expression of eca-mir-128 and quantitative analysis using a housekeeping miRNA (eca-mir-21) is in progress. We have been working on Aim 1 and identified one additional receptor/accessory cellular receptor molecule required for EAV attachment and entry. The preliminary characterization of this cellular protein appears to be vimentin. Further work is in progress to better define the role of this protein in EAV attachment and entry and its role in EAV LTPI in the stallion reproductive tract. We have submitted three abstracts (one to CRAWD 2020 and two to Nido2020 meeting). The Nido2020 meeting was postponed to this year because of the COVID-19 pandemic. We are working with our collaborator at the University of Illinois on acquiring stallions for the in vivo studies described in Aims 2 and 3. Results from these studies will be featured in a webinar hosted by Leica Biosystems on April 27, 2021 and entitled "Automated IHC and ISH for Academic Research."
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Abstracts at National and International Meetings:
1. M. Carossino, P. Dini, A. T. Loynachan, L. Miller, B. Wagner, T. S. Kalbfleisch, I. F. Canisso, P. J. Timoney, and U. B. R. Balasuriya. Host-Virus Interactions Mediating Equine Arteritis Virus Persistence in the Stallion Reproductive Tract. USDA-NIFA AFRI Animal Health, Animal Well-Being and Food Security Awardee Workshop, and the 99th Annual Conference of Research Workers in Animal Diseases (CRWAD) Virtual Meeting 2020.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
2. U. B. R. Balasuriya and M. Carossino. Genomic and Transcriptomic Signatures Associated with Equine Arteritis Virus Persistent Infection in the Stallion. XV International Nidovirus Symposium. Egmond Aan Zee, The Netherlands, June 7th-8th, 2021.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
3. M. Carossino and U. B. R. Balasuriya. Role of MicroRNA eca-mir-128, CXCL16 and other C-X-C Motif Chemokines and Receptors During Equine Arteritis Virus (Alphaarterivirus equid) Persistent Infection. XV International Nidovirus Symposium. Egmond Aan Zee, The Netherlands, June 7th-8th, 2021.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Presentations:
1. Invited presentation (Carossino, M.) - Leica Biosystems: Automating IHC and RNAscope� ISH for diagnostics and academic research . Buffalo Grove, IL. April 27, 2021.
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdoctoral fellows, farm managers and equine industry stakeholders Changes/Problems: The proposed start date for this grant was February 15, 2019, but the funding was released on 11/05/2019 (NIFA [FR] Award Notification 2019-67016-29102; Reference 2020-00387). The delay is due to several reasons. The PI and CO-PI (postdoctoral scholar) joined the School of Veterinary Medicine, Louisiana State University (LSU) on July 01, 2018, and it took approximately nine months to establish their research laboratory. There was a delay in obtaining IACUC approval for funds to be released. One of the postdoctoral scholars took a new job and moved to another university. We plan to recruit a new postdoctoral fellow or a part-time technician. The cost of the automated immunostainer has increased and we need to request a budget reallocation to purchase the Ventana Ultra instrument. Current COVID-19 pandemic may result in further delay in purchasing stallions for experimental inoculation and overall research in the laboratory. The project is currently behind schedule but we plan to pick up pace once we hire the new graduate student. What opportunities for training and professional development has the project provided?1. Dr. Mariano Carossino is a postdoctoral scholar and anatomic pathology resident at the LADDL 2. One graduate student will start in fall, 2020. How have the results been disseminated to communities of interest?Abstracts and presentations What do you plan to do during the next reporting period to accomplish the goals?We will work on all three objectives and try our best to get back on schedule.
Impacts
What was accomplished under these goals?
1. We have established our new research laboratory in room 1066, Louisiana Animal Disease Diagnostic Laboratory (LADDL), Department of Pathobiological Sciences, School of Veterinary Medicine, LSU, Baton Rouge, LA 70803. 2. We have moved all the necessary viruses, archived tissues and reagents from the University of Kentucky to LSU. 3. We have established all necessary immunohistochemical protocols needed for objectives 2 and 3 in the laboratory. 4. We are currently in the process of purchasing an advanced immunohistochemical staining instrument (i.e., Ventana Ultra) for this project. 5. We have collected tissues from three EAV persistently infected stallions and one uninfected stallion to be included in this study. These tissues are very useful as controls in this study. 6. We have selected a new graduate student to work on this project and he will begin on Fall 2020.
Publications
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