Progress 06/01/17 to 05/31/21
Outputs
Target Audience:Scientists and researchers attending the Annual Conference ofResearch Workers inAnimalDiseases (CRWAD). Faculty members, scientists, graduate students, and undergraduate students at the UConn Animal Science Department and Department of Pathobiology and Veterinary Science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph.D. students have been extensively trained in pig macrophage isolation, functional evaluation, PRRSV production and titration, studying signaling pathways that regulate CD163 expression, BiFC assay development to investigate PRRSV-CD163 interaction, and evaluation of compounds to block PRRSV infection. One Ph.D. student has graduated and now doing postdoc research at NIH. This project also provided training opportunities for 5 undergraduate students for laboratory research and molecular and cell biology techniques. This project has helped the PI to develop 2 research projects with Atomwise, Inc., 1 project with the Program in Innovative Therapeutics for Connecticut's Health (PITCH), and 3 projects with the University of Connecticut to establish screening assays and identify small molecules to block the PRRSV infection. How have the results been disseminated to communities of interest?The PD has presented part of the research discovery of using small molecules to fight against PRRSV infection at the 2019 and 2020 CRWAD world conferences. The PD had given two invited presentations of this project research to faculty members, postdocs, graduate and undergraduate students at the UConn Pathobiology and Veterinary Science Department. He also presented the research results to scientists and staff at UConn Technology and Commercialization Services. Two Ph.D. students had presented their project research data at the UConn Animal Science Department seminar series. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. We have established that in PAMs, IL-10/STAT3 pathway significantly stimulates CD163 expression at both transcription and translation levels. We further identified one small molecule (Cryptotanshinone) that significantly blocks IL-10 signaling and inhibits multi-strain of PRRSV infection in PAMs. We further screened and identified 5 additional STAT3 inhibitors that inhibit IL-10 signaling and CD163 expression. 2. We established bimolecular fluorescence complementation (BiFC) assays to evaluate the protein-protein interaction between the CD163 SRCR5 domain and the PRRSV glycoprotein GP2a and GP4. We confirmed the physical interaction between these proteins with our BiFC assays. 3. We have screened a group of small molecules that are predicted to target CD163 using our BiFC assay and identified one compound (B7) that inhibits the interaction between CD163 and PRRSV. We further confirmed that this compound significantly inhibited multi-strain of PRRSV infection in PAMs. Further structural-relationship-analysis revealed that one moiety of this compound and a similar moiety in an analogue of this compound function critically for the inhibition of PRRSV-infection. We further identified 4 B7 analogues that exhibit significant inhibitory effect to PRRSV infection of PAMs. 4. We have conducted a pilot toxicity study of the compound B7 in rats and did not find any acute toxicity in rats upon the drug administration.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Huang C, Bernard D, Zhu J, Dash RC, Chu A, Knupp A, Hakey A, Hadden MK, Garmendia A, Tang Y. 2020 Small Molecules Block the Interaction between Porcine Reproductive and Respiratory Syndrome Virus and CD163 Receptor and the Infection of Pig Cells 2020. BMC Virology Journal.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Huang, C, Zhu, J, Wang, L, Chu, A, Yin, Y, Vali, K, Garmendia, A., Tang, Y. Cryptotanshinone Protects Porcine Alveolar Macrophages from Infection with Porcine Reproductive and Respiratory Syndrome Virus. 2020. Antiviral Research.
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Progress 06/01/19 to 05/31/20
Outputs
Target Audience:Faculty members, scientists, graduate students, and undergraduate students at the UConn Animal Science Department and Department of Pathobiology and Veterinary Science. Scientists and staff at theUConn Technology and Commercialization Services. Changes/Problems:PD is requesting a one year no-cost extension. What opportunities for training and professional development has the project provided?Two Ph.D. students have been extensively trained in pig macrophage isolation, functional evaluation, PRRSV production and titration. They also became expertsto perform analysis of signaling pathways that regulate CD163 expression, and to evaluate the PRRSV permissiveness of pig cells with various signalmodulations. This project also allowed the PI to develop 2 new research initiatives with Atomwise, Inc. and 1 initiative with the Program in Innovative Therapeutics for Connecticut's Health (PITCH) to establish cell based BiFC assays and to identifysmall molecules to interfere with and block the PRRSV infection/replication. How have the results been disseminated to communities of interest?Onestudentpresented part of the research data of using small molecules to preventPRRSV infectionduring the UConn Animal ScienceDepartment seminar. He is scheduled topresent his research results as hisPh.D. dissertation at a campus-wide seminar. The PD has presented the research results to scientists and staff in UConn Technology and Commercialization Services to work on the intellectual disclosure and patent application. What do you plan to do during the next reporting period to accomplish the goals?1. We will compare and determine the PRRSV-infection suppressive effect of the additional IL-10 signaling inhibitors identified by us, and submit a manuscript based on this study. 2. We will complete the revision and publication of the 2 submitted manuscripts, whichare currently under review.
Impacts
What was accomplished under these goals?
1. We have established that in PAMs, IL-10 pathway significantly stimulates CD163 expression at both transcription and translation levels. We further identified one small molecule that significantly blocks IL-10 signaling, and inhibits multi-strain of PRRSV infection in PAMs. 2. We have screened and identified 5 additional compounds that inhibit IL-10 signaling and CD163 expression with greater potency. 3. We have screened a group of small molecules that are predicted to target CD163 using our BiFC assay and identified one compound thatinhibits the interaction between CD163 and PRRSV. We further confirmed that this compoundsignificantly inhibited multi-strain of PRRSV infection in PAMs. Further structural-relationship-analysis revealed that one moielty ofthis compound and a similar moiety inan analogue of this compound function critically for the inhibition of PRRSV-infection. 4. A U.S. provisional patent related to the thirdstudy is in process.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Huang, C, Zhu, J, Wang, L, Chu, A, Yin, Y, Vali, K, Garmendia, A., Tang, Y. Cryptotanshinone Protects Porcine Alveolar Macrophages from Infection with Porcine Reproductive and Respiratory Syndrome Virus. 2020. Antiviral Research.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Huang C, Bernard D, Zhu J, Dash RC, Chu A, Knupp A, Hakey A, Hadden MK, Garmendia A, Tang Y. 2020 Small Molecules Block the Interaction between Porcine Reproductive and Respiratory Syndrome Virus and CD163 Receptor and the Infection of Pig Cells 2020. BMC Virology Journal.
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Progress 06/01/18 to 05/31/19
Outputs
Target Audience:Faculty members, scientists, graduate students, and undergraduate students at the UConn Animal Science Departmentand Department of Pathobiology and Veterinary Science.Scientists and researchersattending the 99thCRWAD world conference in Chicago. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph.D. students became experts in pig macrophage isolation, functionalevaluation, PRRSV production and titration. They were also trained extensively to perform analysis ofsignaling pathways that regulateCD163 expression, and to testusing a variety of approaches to evaluatePRRSV permissivenessof the pigcells modulated. This project further allowed the PI to develop new research initiatives and obtain support fromAtomwise, Inc. and the Program in Innovative Therapeutics for Connecticut's Health (PITCH) to establish cell based assays and identify new small molecules to prevent PRRSV infection. How have the results been disseminated to communities of interest?The PI presented part of the research data of using small molecules to fight against PRRSV infection at the 99th CRWAD world conference in Chicago. One Ph.D. studentalsopresented the project research data duringthe UConn Animal Science Department seminar series. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the evaluation of small compounds for their potential effect in inhibiting PRRSV infection.
Impacts
What was accomplished under these goals?
1. We have confirmed that similar asin human macrophages, in PAM cells, IL-10 pathwaysignificantly stimulates CD163 expression at both transcription and translation levels. We further identified one small molecule that significantly inhibits IL-10 stimulated and basal level expression of CD163 in PAMs, andverified its inhibition ofPRRSV infection of PAMs in vitro. 2. We also identified some other mechanisms thatinhibitCD163 expression, and are working on identifying additional small molecularcompounds that canprevent PAM cells from PRRSV infection. 3. A U.S. provisional patent related to this study is in process.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Tang, Y. A compound Protects Porcine Alveolar Macrophages from Infection with Porcine Reproductive and Respiratory Syndrome Virus; under review: Antiviral Research.
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Progress 06/01/17 to 05/31/18
Outputs
Target Audience:Faculty members, scientists, graduate students, and undergraduate students at the UConn Animal Science Departmentand Department of Pathobiology and Veterinary Science. Scientists at the UConn Center forLicensing and Commercialization Development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project allowed the PI and two Ph.D. students to gain more experience in pig macrophage isolation, functionalevaluation, PRRSV production and titration. Under the PI's guidance, one Ph.D. student wastrained extensively to assay andanalyze the signaling pathways in PAMs that regulateCD163 expression, and testing the effect toPRRSV-permissivenessof the pigcellsby modulating the signalingpathway activities. This project further helped the PI to develop new research initiatives and obtain support fromAtomwise, Inc., the Program in Innovative Therapeutics for Connecticut's Health (PITCH), and UConn Faculty Research Excellence program to identifysmall molecules that canprevent PRRSV infection. How have the results been disseminated to communities of interest? The PI gave an invited presentation to faculty members, postdocs, graduate and undergraduate students at the UConn Pathobiology and Veterinary Science Department. One Ph.D. student from thePI's lab alsopresented the project research datain the UConn Animal Science Department seminar series. What do you plan to do during the next reporting period to accomplish the goals? We plan to complete the evaluation of STAT3 and GR activity regulated CD163 expression in PAMs, determine the effect of their inhibitors in inhibiting CD163 expression and PRRSV infection to pig cells. We further plan to determine the combined effect of STAT3 and GR inhibition forCD163 expression and PRRSV-acceptability in PAMcells.
Impacts
What was accomplished under these goals?
Objective 1. We have established primary PAMs from young and adult pig lungs. We confirmed that similar asin human macrophages, in PAM cells, IL-10 significantly stimulates STAT3 activationand CD163 expression at both transcription and translation levels. We further evaluated inhibiting the IL-10 effector- STAT3 with shRNAs. We first designed shRNAs against pig STAT3. However, the effect of STAT3 knockdownon CD163 expression is not obvious. We suspect that this is due to the abundance of STAT3 mRNA in cells, which leaves considerable level of STAT3 expression after shRNA-mediated knockdown.We are currently evaluatingsmall chemicals to inhibit STAT3. We have obtainedthree STAT3 inhibitors based on literature,evaluatedtheir dosage cytotoxicity to PAMs, and determined their optimal concentrationtoinhibit STAT3 in PAMs. Wearecurrently determining their inhibitory role to CD163 expressionand PRRSV infection-inhibitory effectto pig cells. Objective 2. We also found that the stimulating the glucocorticoid receptor (GR) significantly activatesCD163 expression in PAMs.We designed shRNAs against pig GR. However, similar as STAT3, the effect of GR knockdownon CD163 expression is not obvious. We also suspect that this is due to limited GR knockdown in PAMs due to its abundance in mRNAs.Currently we are evaluatingsmall chemicals to inhibit GR in PAMs.We have obtainedthree GR inhibitors based on literature, and have determinedtheir dosage cytotoxicity to PAMs. We are in the process ofdeterminingthe optimal concentration ofthem toinhibit STAT3 in PAMs. We will then testthe effect of theseGR inhibitors for CD163 expression and PRRSV infection to pig cells.
Publications
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