The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation

<div><p>Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4⁺CD25⁺Foxp3⁺ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15–21, 42–48 and 88–94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.</p></div

Role of baculovirus-expressed proteins GP5, M and N of HP-PRRSV in induction of Tregs proliferation.

<p>(A) Purified recombinant GP5, M and N protein expressed in baculovirus. (B) Percentage of Foxp3⁺ cells in the gated CD4⁺ CD25⁺ subpopulations of GP5, M and N. (C) Concentrations of TGF-β in supernatants of 3-day co-cultures. (D) Concentrations of IL-10 in supernatants of 3-day co-cultures. Data came from three independent experiments. Data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001).</p

Effects of different PRRSV strains on Tregs induction.

<p>(A) Percentage of Foxp3⁺ cells in the gated CD4⁺ CD25⁺ subpopulations of HP-PRRSV and C-PRRSV. (B) Concentrations of TGF-β in the supernatants of 3-day co-cultures. (C) Concentrations of IL-10 in supernatants of 3-day co-cultures. Data came from three independent experiments. Data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05, **<i>P</i>< 0.01 and ***<i>P</i><0.001).</p

Induction of Tregs from lymphocytes co-cultured with PRRSV-infected MoDCs.

<p>(A) Representative flow cytometry profile of lymphocytes following 3 days co-culture of PBMCs with PRRSV-infected MoDCs (upper layer), and only culture with PRRSV-infected PBMCs (lower layer). (B) Percentage of Foxp3⁺ cells in the gated CD4⁺CD25⁺ subpopulations of PBMCs co-cultured with PRRSV-infected MoDC. (C) Percentage of Foxp3⁺ cells in the gated CD4⁺ CD25⁺ subpopulations of PRRSV-infected PBMCs alone. (D) Sorted CD4⁺CD25^high cells. (E) Percentage suppression at the indicated Tregs: peripheral lymphocyte ratios. The lymphocytes exposed to Marc-145 lysate-treated MoDCs were used as negative control. The percentage of suppression was calculated as follows: % suppression = 100 × [1 − (% proliferation w/PRRSV/ % proliferation w/mock)] [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138772#pone.0138772.ref035" target="_blank">35</a>]. Data from three independent experiments. All data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05 and **<i>P</i>< 0.01).</p

15N and 46R in HP-PRRSV N protein were key amino acids in Tregs induction.

<p>(A) Alignment of amino acid sequences of N protein of PRRSV BB0907, S1 and other representative strains. (B) Percentage of Foxp3⁺ cells in the gated CD4⁺CD25⁺ subpopulations of three pairs of synthetic peptides. (C) Additivity assay of 3 and 7 peptides in Treg induction. (D) Concentrations of IL-10 in supernatants of 3-day co-cultures. (E) Concentrations of TGF-β in supernatants of 3-day co-cultures. Data came from three independent experiments. Data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001).</p

Effects of N protein of different virulent PRRSV strains on Tregs induction.

<p>(A) Percentage of Foxp3⁺ cells in the gated CD4⁺CD25⁺ subpopulations of BB-N and S1-N. The BB-M was negative control. (B) Concentrations of TGF-β in supernatants of 3-day co-cultures. (C) Concentrations of IL-10 in supernatants of 3-day co-cultures. Data from three independent experiments. Data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001).</p

Effects of the mutant recombinant PRRSV strains on Tregs induction.

<p>(A) Plaque morphology assay of the recombinant mutant strains. (B) Growth kinetics of the recombinant mutant strains. (C) Percentage of Foxp3⁺ cells in the gated CD4⁺CD25⁺ subpopulations of the recombinant viruses. (D) Concentrations of TGF-β in the supernatants of 3-day co-cultures. (E) Concentrations of IL-10 in the supernatants of 3-day co-cultures. All data are represented as the means ± standard deviation of three independent experiments, and data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05 and **<i>P</i><0.01).</p

Effects on Tregs induction of synthetic peptides of N protein of HP-PRRSV.

<p>(A) Percentage of Foxp3⁺ cells in the gated CD4⁺CD25⁺ subpopulations of 16 synthetic peptides, which covered the whole length of HP-PRRSV N protein. (B) The dose-dependent manner of three peptides 3, 7 and 12. Data came from three independent experiments. Data analysis was done using one-way ANOVA and significant differences are shown (*<i>P</i><0.05 and **<i>P</i>< 0.01).</p

Additional file 1: Table S1. of Influence of the amino acid residues at 70 in M protein of porcine reproductive and respiratory syndrome virus on viral neutralization susceptibility to the serum antibody

Primer sequences for construction of the subgenomic replicon of PRRSV and site-directed mutagenesis. (DOC 54 kb

Data_Sheet_2_Mechanism of Qihuang needle therapy in the management of tic disorders: a clinical trial protocol.pdf

BackgroundQihuang needle therapy is a newly developed acupuncture therapy to treat tic disorders in clinical practice. However, the mechanism to reduce tic severity remains unknown. Changes in intestinal flora and circulation metabolites are perhaps the potential pathogenesis of tic disorders. As a result, we present a protocol for a controlled clinical trial using multi-omics analysis to probe the mechanism of the Qihuang needle in managing tic disorders.MethodsThis is a matched-pairs design, controlled, clinical trial for patients with tic disorders. Participants will be allocated to either an experimental group or a healthy control group. The main acupoints are Baihui (GV20), Yintang (EX-HN3), and Jueyinshu (BL14). The experimental group will receive Qihuang needle therapy for a month, while the control group will receive no interventions.Expected outcomesThe change in the severity of the tic disorder is set as the main outcome. Secondary outcomes include gastrointestinal severity index and recurrence rate, which will be calculated after a 12-week follow-up. Gut microbiota, measured by 16S rRNA gene sequencing; serum metabolomics, assessed via LC/MS; and serum zonulin, assessed by enzyme-linked immunosorbent assay (ELISA), will be used as biological specimen analysis outcomes. The present study will investigate the possible interactions between intestinal flora and serum metabolites and the improvement of clinical profiles, which may elucidate the mechanism of Qihuang needle therapy for tic disorders.Trial registrationThis trial is registered at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/). Registration number: ChiCTR2200057723, Date: 2022-04-14.</p