MOLECULAR DETERMINANTS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS CELL TROPISM

<b>Project Methods</b><br> Objectives 1: Comparative analysis of PRRSV entry pathways into different cell types.In this objective, we will use a combination of pharmaceutical drugs, and dominant negativesto selectively disrupt distinct endocytic pathways in different cell types: MARC-145, PAMs andPK15-pCD163. The cells will be inoculated with wt-PRRSV or PRRSV-K160I mutants.Different steps of the virus entry pathway including binding, internalization and uncoating willbe comparatively analyzed using confocal microscopy.Objective 2: Identify additional cellular factors required for PRRSV infectionIn this objective, PAMs, PK15-pCD163 and MARC-145 cells will be transduced withlentivirus expressing either Flag-tagged GP2 K160 or Flag-tagged GP2 I160. Proteins will be coimmunoprecipitatedusing antibody against Flag-tag and subjected to proteomic analysis toidentify proteins that differentially interact GP2-K160 or GP2-I160. The expression of thecandidate host proteins will be knockdown or overexpressed, following by infection with wt-PRRSV or PRRSV-K160I mutant to determine their potential roles in PRRSV infection.Objective 3: Determine potential alternative cellular targets for PRRSV replication in pigsOur preliminary data show that thepigs inoculated with the rNCV13-K160I mutant exhibited high levels of viremia although thismutant virus does not replicate in PAMs cultured ex vivo. It is possible that therNCV13-K160I mutant virus might infect different tissue and/or cell targets than the wt-rNCV13.The studies described below are designed to identify the potential alternative tissuesand/or cellular targets for PRRSV replication pigs.PRRSV-seronegative, three-week-old pigswill be separately inoculated intramuscularlywith eitherwt-NCV13, or rNCV13-K60I mutant at the dose of 105.0 TCID50 per pig. At 1-, 4-, 7- and 10-days post infection, 4 pigs in each group will be humanely euthanized. During necropsy, lunglavage will be collected, and frequency of PAMs infected with the virus will be quantified byflow cytometry as described in our previous study. Samples of lung, tonsil, and lymph nodes and several other tissues including heart, liver, spleen,kidney, intestine, and stomach will be collected and fixed in 10% neutral buffered formalinfor Immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH) to detect viral infected cells and cell markers. Aportion of freshly collected PAMs and tissues will be processed for single cell RNA sequencing.