Porcine cis-acting lnc-CAST positively regulates CXCL8 expression through histone H3K27ac.

The chemokine CXCL8, also known as the neutrophil chemotactic factor, plays a crucial role in mediating inflammatory responses and managing cellular immune reactions during viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) primarily infects pulmonary alveolar macrophages (PAMs), leading to acute pulmonary infections. In this study, we explored a novel long non-coding RNA (lncRNA), termed lnc-CAST, situated within the Cxcl8 gene locus. This lncRNA was found to be highly expressed in porcine macrophages. We observed that both lnc-CAST and CXCL8 were significantly upregulated in PAMs following PRRSV infection, and after treatments with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Furthermore, we noticed a concurrent upregulation of lnc-CAST and CXCL8 expression in lungs of PRRSV-infected pigs. We then determined that lnc-CAST positively influenced CXCL8 expression in PAMs. Overexpression of lnc-CAST led to an increase in CXCL8 production, which in turn enhanced the migration of epithelial cells and the recruitment of neutrophils. Conversely, inhibiting lnc-CAST expression resulted in reduced CXCL8 production in PAMs, leading to decreased migration levels of epithelial cells and neutrophils. From a mechanistic perspective, we found that lnc-CAST, localized in the nucleus, facilitated the enrichment of histone H3K27ac in CXCL8 promoter region, thereby stimulating CXCL8 transcription in a cis-regulatory manner. In conclusion, our study underscores the pivotal critical role of lnc-CAST in regulating CXCL8 production, offering valuable insights into chemokine regulation and lung damage during PRRSV infection.

Molecular identification and probiotic potential characterization of lactic acid bacteria isolated from the pigs with superior immune responses.

Lactic acid bacteria (LAB) belong to a significant group of probiotic bacteria that provide hosts with considerable health benefits. Our previous study showed that pigs with abundant LAB had more robust immune responses in a vaccination experiment. In this study, 52 isolate strains were isolated from the pigs with superior immune responses. Out of these, 14 strains with higher antibacterial efficacy were chosen. We then assessed the probiotic features of the 14 LAB strains, including such as autoaggregation, coaggregation, acid resistance, bile salt resistance, and adhesion capability, as well as safety aspects such as antibiotic resistance, hemolytic activity, and the presence or absence of virulence factors. We also compared these properties with those of an opportunistic pathogen EB1 and two commercial probiotics (cLA and cLP). The results showed that most LAB isolates exhibited higher abilities of aggregation, acid and bile salt resistance, adhesion, and antibacterial activity than the two commercial probiotics. Out of the 14 strains, only LS1 and LS9 carried virulence genes and none had hemolytic activity. We selected three LAB strains (LA6, LR6 and LJ1) with superior probiotic properties and LS9 with a virulence gene for testing their safety in vivo. Strains EB1, cLA and cLP were also included as control bacteria. The results demonstrated that mice treated LAB did not exhibit any adverse effects on weight gain, organ index, blood immune cells, and ileum morphology, except for those treated with LS9 and EB1. Moreover, the antimicrobial effect of LR6 and LA6 strains was examined in vivo. The results indicated that these strains could mitigate the inflammatory response, reduce bacterial translocation, and alleviate liver, spleen, and ileum injury caused by Salmonella typhimurium infection. In addition, the LR6 treatment group showed better outcomes than the LA6 treatment group; treatment with LR6 substantially reduced the mortality rate in mice. The study results provide evidence of the probiotic properties of the LAB isolates, in particular LR6, and suggest that oral administration of LR6 could have valuable health-promoting benefits.

Characterization of Rongchang piglets after infection with type 2 porcine reproductive and respiratory syndrome virus strains differing in pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the production and health of pigs and causes severe economic losses to the swine industry worldwide. Different pig breeds have been reported to have different levels of susceptibility to PRRSV, and different PRRSV strains may also influence the infectivity and pathogenicity of the virus. In this study, the susceptibility of Rongchang pigs (a prominent local pig breed in China) to PRRSV infection was thoroughly investigated. Rongchang piglets were exposed to two PRRSV strains: HuN4 (highly pathogenic PRRSV) and SD53-1603 (moderately virulent NADC30-like PRRSV). We observed that Rongchang pigs infected with HuN4 displayed significant clinical manifestations, including fever, reduced body weight, and interstitial pneumonia lesions. Routine blood tests revealed that HuN4-infected pigs exhibited slightly decreased levels of red blood cells, hemoglobin, reticulocytes, and a notable increase in monocytes than control pigs. Additionally, the Rongchang pigs exhibiting severe clinical signs presented a higher neutrophil-to-lymphocyte ratio and a lower lymphocyte-to-monocyte ratio. In contrast, SD53-1603 infection did not cause considerable harm to Rongchang pigs, only resulting in slightly elevated leukocytes and lymphocytes. Furthermore, these two PRRSV strains elicited divergent cytokine responses, such that SD53-1603 infection induced higher levels of TNF-α and IFN-γ, whereas HuN4 infection upregulated IL-1ß. These dissimilarities in clinical symptoms, pathological changes, viremia, cytokine expression, and routine blood indices between HuN4 and SD53-1603 infections are critical in understanding the mechanisms of PRRSV infection and developing rational prevention and control strategies against PRRSV.

Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus-host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections.

Unveiling the long non-coding RNA profile of porcine reproductive and respiratory syndrome virus-infected porcine alveolar macrophages.

BACKGROUND: Long noncoding RNA (lncRNA) is highly associated with inflammatory response and virus-induced interferon production. By far the majority of studies have focused on the immune-related lncRNAs of mice and humans, but the function of lncRNAs in porcine immune cells are poorly understood. Porcine reproductive and respiratory syndrome virus (PRRSV) impairs local immune responses in the lungs of nursery and growing pigs, whereas the virus triggers the inflammatory responses. Porcine alveolar macrophage (PAM) is the primary target cell of PRRSV, thus PRRSV is used as an in vitro model of inflammation. Here, we profiled lncRNA and mRNA repertories from PRRSV-infected PAMs to explore the underlying mechanism of porcine lncRNAs in regulating host immune responses. RESULTS: In this study, a total of 350 annotated lncRNAs and 1792 novel lncRNAs in PAMs were identified through RNA-seq analysis. Among them 86 differentially expressed (DE) lncRNAs and 406 DE protein-coding mRNAs were identified upon PRRSV incubation. GO category and KEGG pathway enrichment analyses revealed that these DE lncRNAs and mRNAs were mainly involved in inflammation- and pathogen infection-induced pathways. The results of dynamic correlated expression networks between lncRNAs and their predicted target genes uncovered that numerous lncRNAs, such as XLOC-022175, XLOC-019295, and XLOC-017089, were correlated with innate immune genes. Further analysis validated that these three lncRNAs were positively correlated with their predicted target genes including CXCL2, IFI6, and CD163. This study suggests that porcine lncRNAs affect immune responses against PRRSV infection through regulating their target genes in PAMs. CONCLUSION: This study provides both transcriptomic and epigenetic status of porcine macrophages. In response to PRRSV infection, comprehensive DE lncRNAs and mRNAs were profiled from PAMs. Co-expression analysis demonstrated that lncRNAs are emerging as the important modulators of immune gene activities through their critical influence upon PRRSV infection in porcine macrophages.

PP2A Facilitates Porcine Reproductive and Respiratory Syndrome Virus Replication by Deactivating irf3 and Limiting Type I Interferon Production.

Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase in mammalian cells, is known to regulate the kinase-driven intracellular signaling pathways. Emerging evidences have shown that the PP2A phosphatase functions as a bona-fide therapeutic target for anticancer therapy, but it is unclear whether PP2A affects a porcine reproductive and respiratory syndrome virus infection. In the present study, we demonstrated for the first time that inhibition of PP2A activity by either inhibitor or small interfering RNA duplexes in target cells significantly reduced their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) infection. Further analysis revealed that inhibition of PP2A function resulted in augmented production of type I interferon (IFN). The mechanism is that inhibition of PP2A activity enhances the levels of phosphorylated interferon regulatory factor 3, which activates the transcription of IFN-stimulated genes. Moreover, inhibition of PP2A activity mainly blocked PRRSV replication in the early stage of viral life cycle, after virus entry but before virus release. Using type I IFN receptor 2 specific siRNA in combination with PP2A inhibitor, we confirmed that the effect of PP2A on viral replication within target cells was an interferon-dependent manner. Taken together, these findings demonstrate that PP2A serves as a negative regulator of host cells antiviral responses and provides a novel therapeutic target for virus infection.