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This study evaluates the role of the late viral protein, pA104R, in African swine fever virus immunosuppression. ASFV-encoded pA104R is a putative histone-like protein that is highly conserved throughout different virulent and non-virulent isolates. Previous studies have demonstrated that pA104R plays a vital role in the ASFV replication cycle and is a potential target for antiviral therapy. Here, we demonstrated that pA104R is a potent antagonist of type I interferon signaling. IFN-stimulated response element activity and subsequent transcription of co-transfected and endogenous interferon-stimulated genes were attenuated by pA104R treatment in HEK-293 T cells. Immunoprecipitation assay and reciprocal pull-down showed that pA104R does not interact directly with STAT1, STAT2, or IRF9. However, pA104R could inhibit IFN signaling by attenuating STAT1 phosphorylation, and we identified the critical amino acid residues (R/H69,72 and K/R92,94,97) involved through the targeted mutation functional assays. Although pA104R is a histone-like protein localized to the nucleus, it did not inhibit IFN signaling through its DNA-binding capacity. In addition, activation of the ISRE promoter by IRF9-Stat2(TA), a STAT1-independent pathway, was inhibited by pA104R. Further results revealed that both the transcriptional activation and recruitment of transcriptional stimulators by interferon-stimulated gene factor 3 were not impaired. Although we failed to determine a mechanism for pA104R-mediated IFN signaling inhibition other than attenuating the phosphorylation of STAT1, these results might imply a possible involvement of epigenetic modification by ASFV pA104R. Taken together, these findings support that pA104R is an antagonist of type I interferon signaling, which may interfere with multiple signaling pathways.

Noncoding RNAs regulate the process of Mycobacterium tuberculosis (M. tb) infecting the host, but there is no simultaneous transcriptional information of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) and the global regulatory networks of non-coding RNA. Rv1759c, a virulence factor, is a member of protein family containing the proline-glutamic acid (PE) in M. tb, which can increase M. tb survival. To reveal the noncoding RNA regulatory networks and the effect of Rv1759c on non-coding RNA expression during M. tb infection, we collected samples of H37Rv- and H37Rv△1759c-infected macrophages and explored the full transcriptome expression profile. We found 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv infection, 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv△1759c infection. We constructed lncRNA/circRNA-miRNA-mRNA regulatory networks during H37Rv and H37Rv△1759c infection. We demonstrated the role of one of the hubs of the networks, hsa-miR-181b-3p, for H37Rv survival in macrophages. We discovered that the expression changes of 68 mRNAs, 92 lncRNAs, 26 circRNAs, and 3 miRNAs were only related to the deletion of Rv1759c by comparing the transcription profiles of H37Rv and H37Rv△1759c. Here, our study comprehensively characterizes the transcriptional profiles in THP1-derived-macrophages infected with H37Rv and H37Rv△1759c, which provides support and new directions for in-depth exploration of noncoding RNA and PE/PPE family functions during the infection process.

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5′-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

Mycoplasma bovis (M. bovis) is an important pathogen of the bovine respiratory disease complex, invading lower respiratory tracts and causing severe pneumonia. However, its molecular mechanism largely remains unknown. Host annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein. The current study sought to determine whether ANXA2 could mediate M. bovis adhesion and invasion thereby affecting its induction of inflammatory response. ANXA2 expression was upregulated in M. bovis-infected bovine lung epithelial cells (EBL), and blocking ANXA2 with an anti-ANXA2 antibody reduced M. bovis adhesion to EBL. Compared with uninfected cells, more ANXA2 was translocated from the cytoplasm to the cell surface after M. bovis infection. Furthermore, RNA interference knockdown of ANXA2 expression in EBL cells resulted in a significant decrease in M. bovis invasion and F-actin polymerization. Next, the transcriptomic study of M. bovis-infected EBL cells with and without ANXA2 knockdown were performed. The data exhibited that ANXA2 knockdown EBL cells had 2487 differentially expressed genes (DEGs), with 1175 upregulated and 1312 downregulated compared to control. According to GO and KEGG analyses, 50 genes potentially linked to inflammatory responses, 23 involved in extracellular matrix (ECM) receptor interaction, and 48 associated with PI3K-AKT signal pathways were upregulated, while 38 mRNA binding genes, 16 mRNA 3′-UTR binding genes, and 34 RNA transport genes were downregulated. Furthermore, 19 genes with various change-folds were selected for qPCR verification, and the results agreed with the RNA-seq findings. Above all, the transcription of two chemokines (IL-8 and CXCL5) and a key bovine β-defensin TAP in IL-17 signaling pathway were significantly increased in ANXA2 knockdown cells. Moreover, ANXA2 knockdown or knockout could increase NF-κB and MAPK phosphorylation activity in response to M. bovis infection. Additionally, ANXA2 knockdown also significantly decreased the CD44 transcripts via exon V3 and V7 skipping after M. bovis infection. We concluded that M. bovis borrowed host ANXA2 to mediate its adhesion and invasion thereby negatively regulating molecular expression essential to IL-17 signal pathway. Furthermore, CD44 V3 and V7 isoforms might contribute to this ANXA2 meditated processes in M. bovis infected EBL cells. These findings revealed a new understanding of pathogenesis for M. bovis infection.

Mycoplasma hyopneumoniae is the primary pathogen of swine enzootic pneumonia and causes great economic losses to the swine industry worldwide. In China, M. hyopneumoniae seriously hinders the healthy development of the native black pigs. To prevent and treat porcine respiratory disease caused by M. hyopneumoniae, the characteristics of M. hyopneumoniae strain ES-2 isolated from Chinese native black pig lungs with gross lesions at post-mortem were studied for the first time in this study. Strain ES-2 cell was round or oval cells and most sensitive to kanamycin. The diameters of most strain ES-2 cells ranged from 0.4 to 1.0 μm with maximum viability of 1010 CCU/ml. Experimental challenge of animals with strain ES-2 showed respiratory disease could be reproduced, with pneumonic lung lesions evident. Comparative genomics analysis identified that 2 genes are specific to pathogenic M. hyopneumoniae strains, which may be predicted to be a molecular marker. These findings suggest that the study on the characteristics of M. hyopneumoniae strain ES-2 will guide the rapid and accurate drug use in the clinic, and develop a theoretical foundation for accurately diagnosing and treating the infection caused by pathogenic M. hyopneumoniae.

Porcine sapelovirus (PSV) is the causative pathogen of reproductive obstacles, acute diarrhea, respiratory distress, or severe polioencephalomyelitis in swine. Nevertheless, the pathogenicity and pathogenic mechanism of PSV infection are not fully understood, which hinders disease prevention and control. In this study, we found that PSV was sensitive to type I interferon (IFN-β). However, PSV could not activate the IFN-β promoter and induce IFN-β mRNA expression, indicating that PSV has evolved an effective mechanism to block IFN-β production. Further study showed that PSV inhibited the production of IFN-β by cleaving mitochondrial antiviral signaling (MAVS) and degrading melanoma differentiation-associated gene 5 (MDA5) and TANK-binding kinase 1 (TBK1) through viral 3Cpro. In addition, our study demonstrated that PSV 3Cpro degrades MDA5 and TBK1 through its protease activity and cleaves MAVS through the caspase pathway. Collectively, our results revealed that PSV inhibits the production of type I interferon to escape host antiviral immunity through cleaving and degrading the adaptor molecules.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to the swine industry in China and has caused enormous losses every year. To monitor the epidemiological and genetic characteristics of PRRSV in South China, 6,795 clinical samples from diseased pigs were collected between 2017 and 2021, and 1,279 (18.82%) of them were positive for PRRSV by RT-PCR detecting the ORF5 gene. Phylogenetic analysis based on 479 ORF5 sequences revealed that a large proportion of them were highly-pathogenic PRRSVs (409, 85.39%) and PRRSV NADC30-like strains (66, 13.78%). Furthermore, 93.15% of these highly-pathogenic strains were found to be MLV-derived. We next recovered 11 PRRSV isolates from the positive samples and generated the whole genome sequences of them. Bioinformatic analysis showed that seven isolates were MLV-derived. Besides, six isolates were found to be recombinant strains. These eleven isolates contained different types of amino acid mutations in their GP5 and Nsp2 proteins compared to those of the PRRSVs with genome sequences publicly available in GenBank. Taken together, our findings contribute to understanding the prevalent status of PRRSV in South China and provide useful information for PRRS control especially the use of PRRSV MLV vaccines.

Tuberculosis is the second cause in infectious diseases leading to human death. Understanding the virulence mechanism is inevitable if the disease needs to be fully cured. Therefore, this study aimed to reveal this mechanism by comparing proteomic profiles of intracellular and extracellular virulent strainM.tband bacille Calmette–Guérin (BCG) from infected THP-1cells. First,M.tband BCG infected THP-1 at MOI 10:1. Twelve hours postinfection, intracellular bacteria ofM.tband BCG were collected, whereas the two bacilli cultured in 7H9 broth media were used as the control. Then four groups of bacilli were subjected to proteomic analysis, and differential proteomic profiles betweenM.tband BCG were comparatively analyzed with bioinformatics tools. As a result, we identified a total of 1,557 proteins. Further, they were divided into four groups for comparison ofM.tbversus BCG under 7H9 culture (shorten as out),M.tbin (intracellular) versusM.tbout, BCG in versus BCG out andM.tbin versus BCG in. BetweenM.tbin versus BCG in, a total of 211 differentially expressed proteins were found. Eight proteins like ESAT-6 distributed in six RDs and some known proteins related to virulence. Besides, five uncharacterized proteins were differentially expressed. Further analysis revealed enriched pathways were associated with glyoxylate and dicarboxylate metabolism pathways. InM.tbout versus BCG out, a total of 144 differential proteins were identified and mainly involved in metabolism pathways. Then, 121 differential proteins in the group ofM.tbin versusM.tbout were enriched in ribosome and oxidative phosphorylation related to adaptation to the host environment. The group of BCG in versus BCG out shared the same trend of different pathways to theM.tbin versusM.tbout. Finally, 42 proteins were identified to be up-regulated only in intracellularM.tbincluding eight RD proteins, whereas 22 up-regulated uniquely in intracellular BCG. Besides, only two proteins (Pks13 and Rv1405c) were commonly up-regulated in intracellularM.tband BCG. Further, some unknown proteins were uniquely up-regulated in the intracellularM.tband BCG. These findings provide valuable data for further exploration of molecular mechanism forM.tbvirulence and BCG immune response.

To reveal functions of novel Mycobacterium tuberculosis (M. tb) proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of M. tb genome in inhibiting the host inflammatory response and the underlying mechanism, using in-vitro and in-vivo experiments. A recombinant M. smegmatis strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1β, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1β, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.

The transcription factor NF-κB plays an important role in modulation of inflammatory pathways, which are associated with inflammatory diseases, neurodegeneration, apoptosis, immune responses, and cancer. Increasing evidence indicates that TRIM proteins are crucial role in the regulation of NF-κB signaling pathways. In this study, we identified TRIM67 as a negative regulator of TNFα-triggered NF-κB activation. Ectopic expression of TRIM67 significantly represses TNFα-induced NF-κB activation and the expression of pro-inflammatory cytokines TNFα and IL-6. In contrast, Trim67 depletion promotes TNFα-induced expression of TNFα, IL-6, and Mcp-1 in primary mouse embryonic fibroblasts. Mechanistically, we found that TRIM67 competitively binding β-transducin repeat-containing protein (β-TrCP) to IκBα results inhibition of β-TrCP-mediated degradation of IκBα, which finally caused inhibition of TNFα-triggered NF-κB activation. In summary, our findings revealed that TRIM67 function as a novel negative regulator of NF-κB signaling pathway, implying TRIM67 might exert an important role in regulation of inflammation disease and pathogen infection caused inflammation.

Bacterial meningitis is a life-threatening infectious disease with high morbidity and mortality worldwide, among which meningitic Escherichia coli is a common Gram-negative pathogenic bacterium causing meningitis. It can penetrate the blood–brain barrier (BBB), invoke local inflammatory responses and consequently disrupt the integrity of the BBB. Interleukin-17A (IL-17A) is recognized as a pro-inflammatory cytokine that is released during meningitic E. coli infection. It has been reported that IL-17A is involved in several pathological tissue injuries. However, the function of IL-17A in BBB breakdown remains rarely discussed. Here, our study found that E. coli-induced IL-17A led to the degradation of tight junction proteins (TJs) and adherens junction proteins (AJs) in human brain microvascular endothelial cells (hBMECs) through inhibiting protease proteinase 3 (PRTN3)/protease-activated receptor 2 (PAR-2) axis, thus increasing the permeability of BBB. In summary, this study uncovered the involvement of IL-17A in regulating BBB integrity and proposed a novel regulatory mechanism, which could be potential therapeutic targets of E. coli meningitis.

Pasteurella multocida generally colonizes mammalian/bird respiratory tracts and mainly causes respiratory disorders in both humans and animals. To date, the effects of P. multocida infection on the respiratory epithelial barriers and molecules in host respiratory epithelial cells in their response to P. multocida infection are still not well-known. In this study, we used newborn pig tracheal epithelial (NPTr) cells as an in vitro model to investigate the effect of P. multocida infection on host respiratory epithelial barriers. By detecting the transepithelial electrical resistance (TEER) values of NPTr cells and the expression of several known molecules associated with cell adherens and junctions, we found that P. multocida infection disrupted the barrier functions of NPTr cells. By performing RNA sequencing (RNA-Seq), we determined 30 differentially expressed genes (DEGs), including the vascular endothelial growth factor A (VEGFA) encoding gene VEGFA, which participated in biological processes (GO:0034330, GO:0045216, and GO:0098609) closely related to epithelial adhesion and barrier functions. These 30 DEGs participated in 22 significant signaling pathways with a p-value < 0.05, including the transforming growth factor (TGF)-beta signaling pathway (KEGG ID: ssc04350), hypoxia-inducible factor 1 (HIF-1) signaling pathway (KEGG ID: ssc04066), epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance (KEGG ID: ssc01521), tumor necrosis factor (TNF) signaling pathway (KEGG ID: ssc04668), and mitogen-activated protein kinase (MAPK) signaling pathway (KEGG ID: ssc04010), which are reported to have roles in contributing to the production of inflammatory factors as well as the regulation of epithelial adhesion and barrier function in other tissues and organisms. The results presented in this study may help improve our understanding of the pathogenesis of P. multocida.

Mycoplasma hyopneumoniae causes swine respiratory disease worldwide. Due to the difficulty of isolating and cultivating M. hyopneumoniae, very few attenuated strains have been successfully isolated, which hampers the development of attenuated vaccines. In order to produce an attenuated M. hyopneumoniae strain, we used the highly virulent M. hyopneumoniae strain ES-2, which was serially passaged in vitro 200 times to produce the attenuated strain ES-2L, and its virulence was evidenced to be low in an animal experiment. In order to elucidate the mechanisms underlying virulence attenuation, we performed whole-genome sequencing of both strains and conducted comparative genomic analyses of strain ES-2 and its attenuated form ES-2L. Strain ES-2L showed three large fragment deletion regions including a total of 18 deleted genes, compared with strain ES-2. Analysis of single-nucleotide polymorphisms (SNPs) and indels indicated that 22 dels were located in 19 predicted coding sequences. In addition to these indels, 348 single-nucleotide variations (SNVs) were identified between strains ES-2L and ES-2. These SNVs mapped to 99 genes where they appeared to induce amino acid substitutions and translation stops. The deleted genes and SNVs may be associated with decreased virulence of strain ES-2L. Our work provides a foundation for further examining virulence factors of M. hyopneumoniae and for the development of attenuated vaccines.

Mycoplasmas are successful pathogens both in humans as well as in animals. In cattle, Mycoplasma bovis (M. bovis) is known to be responsible for serious health complications, including pneumonia, mastitis, and arthritis. However, M. bovis pathogenesis remains unclear. Secreted proteins of M. bovis could influence infection and modify host defense signaling pathways after they enter their extracellular space in the host micro-environment. Therefore, this study was aimed to compare the secretomes of M. bovis HB0801 virulent (P1) and attenuated (P150) strains and identify potential pathogenesis-related secreted proteins and biomarkers. The cells of P1 and P150 strains were grown in pleuropneumonia-like organism medium to log phase and then transferred to phosphate-buffered saline for 2 h. Then, the supernatant was analyzed by using label-free quantitative proteomics, and 477 potential secreted proteins were identified. Combined with the bioinformatics prediction, we found that 178 proteins were commonly secreted by the P1 and P150 strains, and 49 of them were encoded by mycoplasmal core genes. Additionally, 79 proteins were found to have a different abundance between the P1 and P150 strains. Among these proteins, 34 were more abundant and uniquely expressed in P1, indicating a possible association with the virulence of M. bovis. Three differentially secreted proteins, MbovP0145, MbovP0725, and MbovP0174, as well as one equally secreted protein, MbovP0481, as positive control and one protein of inner membrane, MbovP0310, as negative control were, respectively, cloned, expressed, and evaluated for antigenicity, subcellular location, and the secretion nature with their mouse antisera by western blotting and colony immunoblotting assay. Among them, MbovP0145 was confirmed to be more secreted by P1 than P150 strain, highly reactive with the antisera from naturally infected and P1 experimentally infected cattle but not with the P150 vaccinated calves, indicating its potential as a diagnostic antigen. In conclusion, these findings may represent the most extensive compilation of potentially secreted proteins in mycoplasma species and the largest number of differentially secreted proteins between the virulent and attenuated M. bovis strains to date and provide new insights into M. bovis pathogenesis and diagnosis.

Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210–269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αβ-crystallin knockout cell line BoMac-cryab−, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab− induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.

Stress granules (SGs) are the sites of mRNA storage and related to the regulation of mRNA translation, which are dynamic structures in response to various environmental stresses and viral infections. Seneca Valley virus (SVV), an oncolytic RNA virus belonging to Picornaviridae family, can cause vesicular disease (VD) indistinguished from foot-and-mouth disease (FMD) and other pig VDs. In this study, we found that SVV induced SG formation in the early stage of infection in a PKR-eIF2α dependent manner, as demonstrated by the recruitment of marker proteins of G3BP1 and eIF4GI. Surprisingly, we found that downregulating SG marker proteins TIA1 or G3BP1, or expressing an eIF2α non-phosphorylatable mutant inhibited SG formation, but this inhibition of transient SG formation had no significant effect on SVV propagation. Depletion of G3BP1 significantly attenuated the activation of NF-κB signaling pathway. In addition, we found that SVV inhibited SG formation at the late stage of infection and 3C protease was essential for the inhibition depending on its enzyme activity. Furthermore, we also found that 3C protease blocked the SG formation by disrupting eIF4GI-G3BP1 interaction. Overall, our results demonstrate that SVV induces transient SG formation in an eIF2α phosphorylation and PKR-dependent manner, and that 3C protease inhibits SG formation by interfering eIF4GI-G3BP1 interaction.

Mycobacterium tuberculosis (M. tb) can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of M. tb-infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during M. tb infection. This study aimed to investigate the roles of miR-378d in M. tb infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, M. tb infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, M. tb survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1β, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during M. tb infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with M. tb infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during M. tb infection of macrophages, miR-378d was down-regulated and functioned on decreasing M. tb intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1β, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against M. tb infection.

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

Rabies virus (RABV) causes fatal encephalitis in mammals and poses a public health threat in many parts of the world. Vaccination remains the most effective means for prevention and control of rabies. Studies focusing on the mechanism of RABV immunogenicity are necessary for improvement of rabies vaccines. Toll-like receptor 7 (TLR7), an innate receptor sensing single-stranded viral RNA, is important for the induction of innate and adaptive immunity. Our studies revealed that the absence of TLR7 led to a lower antibody production in mice immunized with RABV. It is further found that TLR7 deficiency affected the recruitment of germinal center (GC) B cells and led to lessened GCs formation. Consistently, there were less plasma cells (PCs) and antibody secreting cells (ASC) in TLR7−/− mice than those in wild type (WT) mice, resulting in impaired production of RABV-neutralizing antibodies (VNA). TLR7 deficiency also impaired the generation of memory B cells (MBCs) and the induction of secondary immune responses. Moreover, TLR7 deficiency down-regulated the induction of some cytokines/chemokines, especially IFN-γ, resulting in a Th2-biased antibody production. Overall, our results suggest that TLR7 facilitates the induction of the humoral immunity in response to RABV.

Streptococcus suis serotype 2 is a serious zoonotic pathogen and has attracted worldwide attention since the first human case was reported in Denmark in 1968. Some virulence factors have been reported to be involved in the pathogenesis of the infection caused by Streptococcus suis serotype 2, and then novel strategies to identify some anti-virulence compounds which can effectively inhibit the pathogenic bacterial infection have recently been reported. Suilysin is an essential virulence factor for Streptococcus suis serotype 2 since it creates pores in the target cells membranes, which aids bacterial colonization. The important role of suilysin in the virulence of Streptococcus suis serotype 2 renders it an ideal target for designing novel anti-virulence therapeutics. We find that fisetin, as a natural flavonoid, is a potent antagonist against suilysin-mediated hemolysis. The aim of this study is to evaluate the effect of fisetin on the hemolytic activity of suilysin from Streptococcus suis serotype 2. Fisetin is found to significantly inhibit the hemolytic activity of suilysin. Within the range of effective concentrations, fisetin does not influence the growth of Streptococcus suis serotype 2 and the expression of suilysin protein. In vitro, fisetin effectively inhibits the death of macrophages (J774A.1 and RAW264.7) infected with Streptococcus suis serotype 2 by weakening intracellular bacterial multiplication. Animal model experiment shows that fisetin effectively improves the survival rate of animals infected with Streptococcus suis serotype 2. Our findings suggest that fisetin could be used as an antitoxin against suilysin and be developed into a promising therapeutic candidate for treating Streptococcus suis serotype 2 infection.

Pasteurella multocida is a leading cause of respiratory diseases in many host species. To understand the genetic characteristics of P. multocida strains isolated from different host species, we sequenced the genomic DNA of P. multocida isolated from pigs and analyzed the genetic characteristics of strains from avian species, bovine species, pigs, and rabbits using whole genome sequence (WGS) data. Our results found that a capsular: lipopolysaccharide (LPS): multilocus sequence typing (MLST) genotype A: L1: ST129 (43.75%) was predominant in avian P. multocida; while genotypes B: L2: ST122 (60.00%) and A: L3: ST79 (30.00%) were predominate in bovine P. multocida; genotype D: L6: ST50 (37.50%) in porcine P. multocida; and genotype A: L3: ST9 (76.47%) in rabbit P. multocida. Comparative genomic analysis of P. multocida from different host species found that there are no genes in the P. multocida genome that are specific to any type of host. Phylogenetic analysis using either whole-genome single nucleotide polymorphisms (SNPs) or the set of SNPs present in all single-copy core genes across genomes showed that P. multocida strains with the same LPS genotype and MLST genotype were clustered together, suggesting the combining both the LPS and MLST typing schemes better explained the topology seen in the P. multocida phylogeny.

DExD/H-box helicase 36 (DHX36) is known to be an ATP-dependent RNA helicase that unwinds the guanine-quadruplexes DNA or RNA, but emerging data suggest that it also functions as pattern recognition receptor in innate immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide. Interstitial pneumonia is considered to be one of the most obvious clinical signs of PRRSV infection, suggesting that the inflammatory response plays an important role in PRRSV pathogenesis. However, whether DHX36 is involved in PRRSV-induced inflammatory cytokine expression remains unclear. In this study, we found that PRRSV infection increased the expression of DHX36. Knockdown of DHX36 and its adaptor myeloid differentiation primary response gene 88 (MyD88) by small-interfering RNA in MARC-145 cells significantly reduced NF-κB activation and pro-inflammatory cytokine expression after PRRSV infection. Further investigation revealed that PRRSV nucleocapsid protein interacted with the N-terminal quadruplex binding domain of DHX36, which in turn augmented nucleocapsid protein-induced NF-κB activation. Taken together, our results suggest that DHX36–MyD88 has a relevant role in the recognition of PRRSV nucleocapsid protein and in the subsequent activation of pro-inflammatory NF-κB pathway.

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus (CoV) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. Unlike most CoVs that antagonize type I interferon (IFN) production, previous studies showed that TGEV infection induces IFN-I production both in vivo and in vitro. However, the underlying mechanism(s) remain largely unknown. In this study, we found that TGEV infection significantly facilitated IFN-β production as well as activation of the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor-kappaB (NF-κB) in porcine kidney (PK-15) cells. Screening of TGEV-encoded proteins demonstrated that non-structural protein 14 (nsp14) was the most potent IFN-β inducer and induced IFN-β production mainly by activating NF-κB but not IRF3. Further analysis showed that nsp14 interacted with DDX1, a member of the DExD/H helicase family. Knockdown of DDX1 by specific small interfering RNA (siRNA) significantly decreased nsp14-induced IFN-β production and NF-κB activation. Furthermore, TGEV-induced IFN-β production and IFN-stimulated gene (ISG) expression were decreased in cells transfected with DDX1-specific siRNA, indicating the vital role of DDX1 to TGEV-induced IFN-β responses. In summary, our data revealed a potential coactivator role of host RNA helicase DDX1 to the induction of IFN-β response initiated by TGEV and demonstrated that nsp14 is an important IFN inducer among the TGEV-encoded proteins.

Mycoplasma bovis is an important cause of bovine respiratory disease worldwide. To understand its virulence mechanisms, we sequenced three attenuated M. bovis strains, P115, P150, and P180, which were passaged in vitro 115, 150, and 180 times, respectively, and exhibited progressively decreasing virulence. Comparative genomics was performed among the wild-type M. bovis HB0801 (P1) strain and the P115, P150, and P180 strains, and one 14.2-kb deleted region covering 14 genes was detected in the passaged strains. Additionally, 46 non-sense single-nucleotide polymorphisms and indels were detected, which confirmed that more passages result in more mutations. A subsequent collective bioinformatics analysis of paralogues, metabolic pathways, protein-protein interactions, secretory proteins, functionally conserved domains, and virulence-related factors identified 11 genes that likely contributed to the increased attenuation in the passaged strains. These genes encode ascorbate-specific phosphotransferase system enzyme IIB and IIA components, enolase, L-lactate dehydrogenase, pyruvate kinase, glycerol and multiple sugar ATP-binding cassette transporters, ATP binding proteins, NADH dehydrogenase, phosphate acetyltransferase, transketolase, and a variable surface protein. Fifteen genes were shown to be enriched in 15 metabolic pathways, and they included the aforementioned genes encoding pyruvate kinase, transketolase, enolase, and L-lactate dehydrogenase. Hydrogen peroxide (H2O2) production in M. bovis strains representing seven passages from P1 to P180 decreased progressively with increasing numbers of passages and increased attenuation. However, eight mutants specific to eight individual genes within the 14.2-kb deleted region did not exhibit altered H2O2 production. These results enrich the M. bovis genomics database, and they increase our understanding of the mechanisms underlying M. bovis virulence.

Influenza virus NS2 is well known for its role in viral ribonucleoprotein nuclear export; however, its function has not been fully understood. A recent study showed that NS2 might interact with HIST1H1C (H1C, H1.2). Histones have been found to affect influenza virus replication, such as the H2A, H2B, H3, and H4, but H1 has not been detected. Here, we found that H1C interacts with NS2 via its C-terminal in the nucleus and that H1C affects influenza virus replication. The H1N1 influenza virus replicates better in H1C knockout A549 cells compared to wild-type A549 cells, primarily because of the regulation of H1C on interferon-β (IFN-β). Further studies showed that the H1C phosphorylation mutant (T146A) decreases IFN-β, while H1C methylation mutants (K34A, K187A) increases IFN-β by releasing the nucleosome and promoting IRF3 binding to the IFN-β promoter. Interestingly, NS2 interacts with H1C, which reduces H1C–IRF3 interaction and results in the inhibition of IFN-β enhanced by H1C. In summary, our study reveals a novel function of H1C to regulate IFN-β and uncovers an underlying mechanism, which suggests H1C plays a role in epigenetic regulation. Moreover, our results suggest a novel mechanism for the influenza virus to antagonize the innate immune response by NS2.

UNLABELLED DNA vaccines containing the capsid precursor polypeptide P1 gene of foot-and-mouth disease virus (FMDV) alone or combined with the VP22 gene of bovine herpesvirus 1 (BVP22) as molecular adjuvant were constructed and used for immunization of BALB/c mice. The latter were challenged with FMDV and their humoral as well as cell-mediated immune responses and virus clearance capacity were assayed. Both DNA vaccines elicited specific immune responses, however, the DNA vaccine with the BVP22 adjuvant showed stronger responses and more efficient virus clearance. A stronger Th1 response was indicated by the IgG2a/IgG1 ratio. These results indicate that (i) a DNA vaccine based on FMDV P1 can stimulate significant immune responses and virus clearance and (ii) BVP22 is a potentially useful molecular adjuvant for such a vaccine. KEYWORDS DNA vaccine; foot-and-mouth disease virus; bovine herpesvirus 1.