POTENTIAL OF PRP AS ANTIVIRAL THERAPEUTICS IN PIGS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1024188
• Project No.: NCV-VMAH-0161
• Project Start Date: Aug 18, 2020
• Project End Date: Sep 30, 2021

Project Director
Crisci, EL.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Population Health and Pathobiology

Non Technical Summary
Porcine Respiratory Disease Complex (PRDC) is a multifactorial disease affecting ~30- 70% of pigs upon break-out on a unit. Outbreaks usually occur at 14-20 weeks of age and severity of disease highly depends on management, environmental stressors and pathogen evolution (1). Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and swine influenza virus (SwIV) are key players in PRDC, impairing the normal function of the respiratory immune system. The combination with other viral and bacterial secondary infections can lead to reduced performance, increased medication costs and mortality (2-4).Alternative methods such as natural compounds with antiviral properties could be used to reduce infection and boost immunity as a complementary measure to current vaccination due to their lower secondary effects when compared to synthetic drugs (5).Platelets play an important role in innate immunity, since they degranulate and release antimicrobial peptides (AMPs) when activated (6). Platelet rich plasma (PRP) is an autologous compound obtained from an individual's blood that has recently showed to present potent antimicrobial properties and positive immunological effects (7-9). Dr. Gilbertie has recently described a processing method that isolates the AMPs within PRP and has provided extensive in vitro and in vivo data supporting its antibacterial activity (U.S. Provisional Patent Application Serial No. 62/908,318).Our hypothesis is that treating infected cells with PRP can significantly reduce viral load and boost the immune response.As a preliminary exploration to ensure the feasibility of this project, we collected porcine PRP from whole blood following the processing methods described by Dr. Gilbertie to isolate AMP-rich PRP in horses (10-11).To assess the antiviral effects of PRP, we measured the viral load after infection with SwIV and PRRSV in PRP-treated and untreated cells. The expression of mRNA after infection was tested via RT-qPCR (for both viruses) and flow cytometry (for SwIV). We assessed the antiviral effects of PRP with multiplicity of infection (MOI) ranging from 0.05 to 1, and two different treatments: i) PRP post-infection (virus-PRP) and ii) PRP during and post-infection (virus-PRP+PRP). Preliminary data showed antiviral activity in all tested conditions, for all MOIs and for two time points.Due to the promising results obtained, we have already submitted an invention disclosure (No. 20186) for the porcine BIOPLY and its antiviral activity.Based on these preliminary data, the aim of this project is to assess whether PRP can efficiently decrease viral load in vitro and increase immune response in primary lung immune cells.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3599	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1090 - Immunology;

Keywords

porcine respiratory disease complex

swine

immunology

Goals / Objectives
The aim of this project is to assess whether PRP can efficiently decrease viral load in vitro and increase immune response in primary lung immune cells.

Project Methods
As a preliminary exploration to ensure the feasibility of this project, we collected porcine PRP from whole blood following the processing methods described by Dr. Gilbertie to isolate AMP-rich PRP in horses (10-11).To assess the antiviral effects of PRP, we measured the viral load after infection with SwIV and PRRSV in PRP-treated and untreated cells. The expression of mRNA after infection was tested via RT-qPCR (for both viruses) and flow cytometry (for SwIV).To accomplish the proposed goals, we will perform the same procedures (preliminary data) in different IV subtypes and PRRSV strains and in immortalized porcine tracheal cell line (NPTr, for IVs) (12-13). Then we will use primary cells isolated from porcine respiratory tissues, which will better simulate in vivo conditions, for both viruses.The antiviral effects of PRP will be assessed by measuring viral gene expression (RT-qPCR), viral protein (flow cytometry) and viral progenies (TCID50 method) in uninfected versus infected (PRP-treated and non-treated) cells (14). Finally, we will evaluate the variations in cellular immune responses by measuring cytokine gene expression via RT-qPCR.

Progress 08/18/20 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems:Due to COVID19 all the experiments were interrupted after March 13th 2020. In particular, the animal order to perform the genomic approach was cancelled and library preparation and sequencing were not performed in the proposed timeline. After the shutdown, the activities restarted slowly (June 2020) and shifts had limited the capacity to perform long and time consuming experiments (e.g. immunogenomics). The preparation of the samples for the RNAseq was performed only during March 2021, when we performed fresh lung cell isolation, PRRSV infection and sorting of specific MNP. What opportunities for training and professional development has the project provided?The project was an opportunity for training a graduate student and 2 undergrad students. How have the results been disseminated to communities of interest?An abstract was presented at the American Society for Virology 39th Annual Meeting 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 collected several pig lungs from TAU, SEU animals and isolated lung mononuclear phagocytes (MNP). Subsequently, MNP subsets were isolated using cell magnetic separation, sorting and macrophages adhesion approach. We optimized a sorting strategy using porcine MHCII, CD163, CD172a to obtain three different subpopulations: alveolar macrophages (AM), pulmonary intravascular macrophages (PIM), monocyte-derived dendritic cells (moDC), pool of conventional DC (cDC1+cDC2), monocyte-macrophages (moMAC). We have evaluated the modulation of MNP subpopulations during PRRSV-2 infection with two different strains; NC 1-7-4 (highly pathogenic) and NC 1-3-4 (low pathogenic). We have compared the macrophage adhesion approach with macrophages magnetic separation and we have found no differences in outcome, so both approaches can be used in vitro. We have characterized cytokine expression (12 different cytokines) and viral transcripts in lung MNP subsets infected with different PRRSV-2 strains (NC 1-7-4, NC 1-3-4, MLV). We have generated several PRRSV-2 stocks, purchased the PRRSV-2 strain VR-2332 and received from a collaborator the PRRSV-1 strain for future comparison. We have performed a whole genome sequencing of our field isolates (NC 1-7-4 and NC 1-3-4) using Illumina technologies and UNC genomic facility. The analysis of the sequences is in process. We have standardized and performed Seahorse assays using Cell Mito Stress kit on macrophages infected with PRRSV-2 at different time points and MOIs. Seahorse assays was performed with all the PRRSV-2 strains in alveolar macrophages and mitochondrial function was differently modulated depending on the strain. We started to generate the samples for RNAseq in a pilot test. We have collected MNP from 2 animals housed in LAR, then infected in vitro with PRRSV-2 NC 1-3-4 and NC 1-7-4 (MOI=1 for 12h) and mock-infected. After infection, different subsets were sorted and total RNA isolated and measured. The samples were prepared for the future RNAseq. Five additional samples for RNAseq were collected during march 2021. We have collected sorted MNP infected with NC 1-7-4 and NC 1-3-4 and prepared RNA for the library preparation.

Publications