Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
Animal Science
Non Technical Summary
Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is the most economically devastating disease affecting swine industry worldwide. The sows with PRRS exhibit reproductive problems such as abortion, premature birth, and stillbirth. All PRRSV infected pigs develop symptoms such as coughing, difficulty breathing, inappetence, poor weight gain, with distress complicated by secondary infections provoking more serious diseases and mortality. Because of the high genetic heterogeneities of PRRSV, currentlyno broadly effective vaccine is availableagainst PRRS. Based on our recent studies, we plan 1) to evaluate the inhibitory effect on PRRSV infection in pigs by a natural compound (and its herb form) that can downregulate the PRRSV specific receptor expression in pig cells to inhibit PRRSV-infection, and to evaluate the derivatives and analogues of this compound to inhibit PRRSV-infectionin vitro; 2) to develop a lead compound from the derivatives/analogues of another small moleculethat blocks the interaction of PRRSV with its receptor and therefore PRRSV infection, and evaluate thein vivotoxicity and efficacy of this lead compound on inhibiting PRRSV infection in pigs; and 3) to perform high-throughput screening to identify additional compounds that block PRRSV binding to its receptor and therefore the viral infection. The data generated from this project will identify novel veterinary medications to block PRRSV infection in pigs. This study will also generatenew knowledge on the PRRSV specific receptor expression and its interaction with PRRSV,to facilitate the identification and test of new therapeutics to curtail the PRRS panzootic.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Thegoal of this project is to identify phytoceuticals or synthetic compounds for prophylactic or therapeutic use (either alone or as adjunct to vaccination programs) for porcine reproductive and respiratory syndrome (PRRS) in pigs.Theoverall objectives are1)to determine the inhibitory effect of a specific natural compoundand as a more cost-effective approach, its herb formon PRRSV infectionin vivo, and identify additional lead compounds from the derivatives/analogues of this compoundon PRRSV infection ofporcine alveolar macrophages (PAMs);2)to develop a lead compound from a specific synthetic compoundand its derivatives/analogues and characterize this compound for toxicity and efficacy on inhibiting PRRSV infectionin vivo; and3)to perform high-throughput (HTP) screening on a small chemical library of 150,000 compounds, to identify additional compounds that block the PRRSV-CD163 interaction and PRRSV infection of PAMsin vitro.
Project Methods
For objective 1, we will (A) Determine theIn VivoInhibitory Effects of the Specific Natural Compound and Its Herb Form on CD163 Expression and PRRSV Infection inPigs. This will be done in 3 to 4-week old cross-bred male and female PRRSV-free piglets divided in 4 groups. Group 1 will be non-infected, non-treated control. For other groups, pigs will be inoculated on day 0 with PRRSV strain VR-2332 by oronasal inoculation. Group 2 will serve as the infection control and will be treated orally with 225 mL vehicle once daily for 7 days following PRRSV inoculation. Groups 3 and 4 will be treated for 7 days with the natural compound suspended in vehicle, with a low and high daily oral dosage, respectively. Groups 5 and 6 will be treated daily for 7 days with the herb powderin vehicleat low and high dose, respectively. Thepigs will be evaluated for 2 weeks after viral inoculation, based on their clinical response. Blood viremia will be measured by quantitative reverse transcription PCR (qRT-PCR). Plasma serial diluents will be co-cultured with MARC-145 cells to monitor cytopathogenic effect forPRRSV titration. The compound plasma concentration will be measured using Liquid Chromatography-Mass Spectrometry (LC-MS). On day 14, pigs will be euthanized and post-mortem examinations performed. The lungs will be examined macroscopically for signs of inflammation and PRRSV infection, weighted, and bronchoalveolar lavage performed. Alveolar macrophages will be subject to RNA extraction for qRT-PCR detection of CD163 expression and PRRSV RNA level. Tissue samples will be collected from eachlung lobe(7 plus 2 additional sections from the large lobes), tracheobronchiallymph nodes, spleen, andinguinal lymph nodes, and will be frozen or fixed in 10% neutral bufferedformalin. Fixed tissues will be sectioned, and stained withhematoxylinandeosinfor histologic evaluation.The lungs will be scored microscopically on 9 sections per pig for lesions. Viral loads will be measured in frozen tissues by qRT-PCR. To evaluate any potential toxic effect by the treatment, we will also examine the pig brain, lung, heart, kidney and liver sections microscopically. Whole blood cell count and complete blood chemistry panel will be analyzed. We will also (B) Collect Primary PAMs and Determining the PRRSV-Susceptibility forIn VitroAssay, and Identify Additional Lead Compounds from the Derivatives/Analogues of the Natural Compound that Potently Inhibiting CD163 Expression and PRRSV InfectionIn Vitro. Atomwise, Inc. will support our effort to identify any derivatives that may exhibit superior potency and bioavailability than this natural compound. They will perform SAR-optimization to design and produce derivatives to further improve the solubility, bioavailability and potency. We will then evaluate all these compounds for their aqueous solubility, cytotoxicity, and inhibitory effects on CD163 expression in PAMs. We will further determine the inhibitory effect of these derivatives/analogues for PRRSV-susceptibility in PAMs. Up to 3 derivative/derivatives with superior solubility and CD163 inhibitory potency will be selected based on the LC50and IC50values. LV and VR-2332 strains of PRRSV (MOI = 0.1-1) will be used for PRRSV infection separately. The PRRSV-susceptibility of treated PAMs will be evaluated by qRT-PCR, immunostaining/FACS, and plaque assay for viral RNA and protein expression, and viral production. We will identify the lead compounds with the greatest potency to inhibit PRRSV-infection. For objective 2, we will (A) Identify A Lead Compound from the Specific Synthetic Compound and Its Analogues/Derivatives with the Most Potent Inhibition on PRRSV-Infection of PAMsIn Vitro. We will 1) determine the maximum aqueous (saline) solubility of this compound and any lead compounds identified below. 2) Based on the compound structure, we have found five additional commercially available analogues from the NCBI PubChem database. Also, Atomwise Inc. will provide ~20 additional analogues for us to test. 3) Atomwise Inc. will perform SAR-optimization based on the compound structure and the 3-D protein structure of CD163-SRCR5, to generate new derivatives for further improvement of compound solubility, bioavailability and potency. We will determine the solubility, LC50, and IC50for all analogues/derivatives. Using the bimolecular fluorescence complementation (BiFC) and PRRSV-infection assays developed by us, we will compare the potency of these compounds for PRRSV inhibition, and identify the lead compound with superior aqueous solubility and potency forin vivostudies. We will then (B) Evaluate the Toxicity of the Lead Compound in Pigs. We will first perform a 7-day pilot study in 4-week old piglets via intramuscular (IM) injection to evaluate the acute toxicity of the selected lead compound and determine the appropriate dosage in pigs for efficacy studies. We will observe clinical symptoms, collect data, and will examine the pig brain, lung, heart, kidney and liver sections microscopically after euthanasian. Whole blood cell count and complete blood chemistry panel will also be analyzed. We will then (C) Evaluate the Inhibition of PRRSV Infection in Pigs by the Lead Compound. The maximum non-toxic dose of the lead compound as determined above will be chosen to evaluate thein vivoinhibition of PRRSV infection. The compound will beIMadministered after PRRSV inoculation. The experiment duration and methodology for evaluation of compound efficacy against PRRSV challenge will be similar as described in objective 1 (A). For objective 3, we will conduct high throughput (HTP) screening using the BiFC assays in 384-well-plate format. We will utilize an in-house pre-selected small chemical library with 150,000 diverse chemotypes and pharmacophores (ChemBridge) maintained at the UConn HTP Facility. For BiFC assay signal detection, the 293T cells will be fixed at 24 h after compound treatment with formaldehyde (4%) and counterstained with Hoechst 33342 in PBS. Bright field, Hoechst nuclear stain, and fluorescence images of cells in each well will be acquired under a scanning microscope capable of automated imaging of 96 to 384-well-plates. The positive inhibition will be identified by fluorescent intensity analysis, and confirmed by examining the fluorescent images. Up to 20 positive compounds identified here with the most significant inhibitory effects will be further evaluated, by testing the cytotoxicity of these compounds in PAMs, and determining theirin vitroinhibitory effects on PRRSV infection of PAMs similarly as described in objectives 1 and 2.