Porcine alveolar macrophage (PAM) is one of the primary cellular targets for PRRSV, but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystanders PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP) and GFP + (PRRSV infected) and GFP – (bystander) cells were sorted for RNA-sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP + and 0.06% reads from GFP – PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP + as compared to GFP – PAMs. Importantly, negative immune regulators including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3), and T-cell exhaustion markers (PD-L1, PD-L2, IL10, IDO1, and TGFB2) were highly upregulated in GFP + cells as compared to GFP – cells. By using in situ hybridization assay, RNA transcripts of TNF and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. Importance PRRSV is widespread in many swine producing countries, causing substantial economic loses to the swine industry. PAM is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAM from an acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV-GFP strain to infect pigs and sorted infected- and bystander- PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of and NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

Both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (PRRSV) strains can establish persistent infection in lymphoid tissues of pigs. To investigate the mechanisms of PRRSV persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated PRRSV strain at 46 days post infection. A total of 6404 differentially expressed genes (DEGs) were detected of which 3960 DEGs were upregulated and 2444 DEGs were downregulated. Specifically, genes involved in innate immune responses and chemokines and receptors associated with T-cell homing to lymphoid tissues were down regulated. As a result, homing of virus-specific T-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific T-cell in lymphoid tissue than in peripheral blood. Genes associated with T-cell exhaustion were upregulated. Likewise, genes involved in the anti-apoptotic pathway were upregulated. Collectively, the data suggested that the live-attenuated PRRSV strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, T-cell homing, and preventing cell apoptosis.

No abstract

A molecular and cellular understanding of the retrograde axonal transport process that underlies the neuroinvasive properties of the alphaherpesviruses is established from studies of herpes simplex virus and pseudorabies virus. The degree to which this phenotype is conserved in other related viruses has largely not been examined.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism for macrophages and CD163 is a key receptor for infection. In this study, the PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, and two plaque-clones (C1 and C2) were randomly selected for further analysis. The C1 virus nearly lost the ability to infect porcine alveolar macrophages (PAMs), as well as porcine kidney cells expressing porcine CD163 (PK15-pCD163), while the C2 virus replicates well in these two cell types. Pretreatment of MARC-145 cells with an anti-CD163 antibody nearly blocked C1 virus infection, indicating that the virus still required CD163 to infect cells. The C1 virus carried four unique amino acid substitutions: three in the nonstructural proteins and a K160I in GP2. The introduction of an I160K substitution in GP2 of the C1 virus restored its infectivity in PAMs and PK15-pCD163 cells, while the introduction of a K160I substitution in GP2 of the low-passaged, virulent PRRSV strain NCV13 significantly impaired its infectivity. Importantly, pigs inoculated with the rNCV13-K160I mutant exhibited lower viremia levels and lung lesions than those infected with the parental rNCV13. These results demonstrated that the K160 residue in GP2 is one of the key determinants of PRRSV tropism.