Mitochondrial dysfunction in PRRSV-2 infected macrophages

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is one of the most economically devastating viruses for the global swine industry. PRRSV has a known tropism for lung macrophages, where it causes impaired immune responses. This study evaluated the metabolic and immune profiles of primary porcine alveolar macrophages (PAM) and pulmonary intravascular macrophages (PIM) infected with different strains of PRRSV-2 isolated from North Carolina (NC) pig herds (NC134, NC18-9-7 referred to as NC174, NC20-1 referred to as NC144), and VR2232, a PRRSV-2 prototype strain. Primary enriched mononuclear phagocytes were infected ex vivo with NC134 and NC174, sorted, and the total RNA was used for a transcriptomic approach; additionally, gene expression was further validated by RT-qPCR and NanoString technology. Complementary functional assays with additional NC strains were used to further investigate the oxidative stress, mitochondrial and metabolic dysfunction induced by PRRSV-2 infection. PAM infected with both NC PRRSV-2 strains NC174 and NC134 showed similar transcriptomic profiles during the early stage of infection, with downregulation of genes involved in the oxidative phosphorylation and electron transport chain pathways. PIM infected with both NC174 and NC134 strains showed limited alteration in the transcriptomic profiles compared to uninfected cells. The genetic reprogramming matched the PRRSV-2 induced mitochondrial impairment observed in functional assays performed with Seahorse technology. Mitochondrial respiration displayed slightly different profiles between PIM and PAM infected with the different PRRSV-2 strains, with PAM showing a more substantial decrease in mitochondrial fitness compared to control cells. When reactive oxygen species (ROS) and nitric oxide (NO) production were evaluated, no differences were observed between PRRSV-2 infected PAM and PIM and control cells. These results provide valuable insights into the pathogenetic mechanism of different North Carolina PRRSV-2 strains, focusing on the alteration of mitochondrial function in lung macrophages during early infection and highlighting differences in lung macrophages responses to distinct PRRSV-2 strains Overall design: Lung bronchoalveolar lavage (BAL) and parenchyma (PAR) samples were isolated from two male and two female animals between 8-12 weeks of age were used. For each pig, thirty million enriched mononuclear pahgocytes (MNP) from both BAL and PAR were infected with NC134 and NC174 at MOI=1 or left uninfected in 50 ml falcon tubes for 12h. Infections were synchronized and after 12h cells were washed and stained with a five-color flow cytometry staining to sort AM, PIM, monocyte derived dendritic cells (moDC) and classical DC (cDC), PAM were sorted from BAL samples and PIM, cDC and moDC were sorted from PAR samples. After sorting, approximately 300,000 PAM and 100,000 PIM were collected for each condition.A range between 63,000 and 100,000 were collected for moDC and cDC. Total RNA was obtained from 63,000-100,000 sorted PAM, PIM, moDC, cDC of four pigs (two females and two males) using the PicoPure RNA Isolation Kit (Thermo Fisher, KIT0204) following manufacturer's instructions. RNA quantity was determined using a Nanodrop 1000 (Thermo Fisher) and an Agilent Bioanalyzer (Agilent), with RIN values ranging from 7.1-9.6 and concentrations ranging from 0.11-21.61 ng/uL. RNA-seq libraries were generated from 1ng of total RNA per sample, using a NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs) following the manufacturer's instructions. Amplified cDNA quality and quantity were assessed using an Agilent Technologies 2200 TapeStation with high sensitivity D5000 tape. For library preparation, 2ng of amplified cDNA per sample were used. Library quality was assessed using an Agilent Technologies 2200 TapeStation with D1000 tape. Libraries were quantified by RT-qPCR using a NGS Library Quantification kit (Takara Bio) following the manufacturer's instructions. A total of 47 cDNA libraries were diluted to 10 nM, pooled, and submitted to Novogene for 150-bp paired-end sequencing on a NovaSeq 6000 S4 lane (Illumina).