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Abstract The surge in advanced imaging techniques has generated vast biomedical image data with diverse dimensions in space, time and spectrum, posing big challenges to conventional compression techniques in image storage, transmission, and sharing. Here, we propose an intelligent image compression approach with the first-proved semantic redundancy of biomedical data in the implicit neural function domain. This Semantic redundancy based Implicit Neural Compression guided with Saliency map (SINCS) can notably improve the compression efficiency for arbitrary-dimensional image data in terms of compression ratio and fidelity. Moreover, with weight transfer and residual entropy coding strategies, it shows improved compression speed while maintaining high quality. SINCS yields high quality compression with over 2000-fold compression ratio on 2D, 2D-T, 3D, 4D biomedical images of diverse targets ranging from single virus to entire human organs, and ensures reliable downstream tasks, such as object segmentation and quantitative analyses, to be conducted at high efficiency.

Abstract Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report a high-throughput Bessel oblique plane microscopy (HBOPM) platform capable of 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity against cancer cells. The HBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells are captured and comprehensively analyzed; these events include the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identify the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.

Abstract Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.

Abstract Background Protein phosphatases type 2C (PP2C) are heavily involved in plant growth and development, hormone-related signaling pathways and the response of various biotic and abiotic stresses. However, a comprehensive report identifying the genome-scale of PP2C gene family in ginger is yet to be published. Results In this study, 97 ZoPP2C genes were identified based on the ginger genome. These genes were classified into 15 branches (A-O) according to the phylogenetic analysis and distributed unevenly on 11 ginger chromosomes. The proteins mainly functioned in the nucleus. Similar motif patterns and exon/intron arrangement structures were identified in the same subfamily of ZoPP2Cs. Collinearity analysis indicated that ZoPP2Cs had 33 pairs of fragment duplicated events uniformly distributed on the corresponding chromosomes. Furthermore, ZoPP2Cs showed greater evolutionary proximity to banana’s PP2Cs. The forecast of cis-regulatory elements and transcription factor binding sites demonstrated that ZoPP2Cs participate in ginger growth, development, and responses to hormones and stresses. ZoERFs have plenty of binding sites of ZoPP2Cs, suggesting a potential synergistic contribution between ZoERFs and ZoPP2Cs towards regulating growth/development and adverse conditions. The protein–protein interaction network displayed that five ZoPP2Cs (9/23/26/49/92) proteins have robust interaction relationship and potential function as hub proteins. Furthermore, the RNA-Seq and qRT-PCR analyses have shown that ZoPP2Cs exhibit various expression patterns during ginger maturation and responses to environmental stresses such as chilling, drought, flooding, salt, and Fusarium solani. Notably, exogenous application of melatonin led to notable up-regulation of ZoPP2Cs (17/59/11/72/43) under chilling stress. Conclusions Taken together, our investigation provides significant insights of the ginger PP2C gene family and establishes the groundwork for its functional validation and genetic engineering applications.

Abstract Interrogation of subcellular biological dynamics occurring in a living cell often requires noninvasive imaging of the fragile cell with high spatiotemporal resolution across all three dimensions. It thereby poses big challenges to modern fluorescence microscopy implementations because the limited photon budget in a live-cell imaging task makes the achievable performance of conventional microscopy approaches compromise between their spatial resolution, volumetric imaging speed, and phototoxicity. Here, we incorporate a two-stage view-channel-depth (VCD) deep-learning reconstruction strategy with a Fourier light-field microscope based on diffractive optical element to realize fast 3D super-resolution reconstructions of intracellular dynamics from single diffraction-limited 2D light-filed measurements. This VCD-enabled Fourier light-filed imaging approach (F-VCD), achieves video-rate (50 volumes per second) 3D imaging of intracellular dynamics at a high spatiotemporal resolution of ~180 nm × 180 nm × 400 nm and strong noise-resistant capability, with which light field images with a signal-to-noise ratio (SNR) down to -1.62 dB could be well reconstructed. With this approach, we successfully demonstrate the 4D imaging of intracellular organelle dynamics, e.g., mitochondria fission and fusion, with ~5000 times of observation.

Weaning is undoubtedly one of the most crucial stages in the growth and development of all mammalian animals, including donkey foals. Weaning is a dynamic and coordinated process of the body, which is closely associated with the health, nutrition, and metabolism of the host. Many studies have shown that the intestinal microbiota and serum metabolites of mammals exhibit different changes during lactation, weaning, and postweaning. However, the alterations in serum metabolites in donkey foals before and postweaning and the correlation between serum metabolites and intestinal microbiota are largely unknown. This study is based on the fecal 16S rRNA and serum metabolomes of Dezhou donkey foals. In total, 10 samples (fecal and serum) were collected during the following three stages: before weaning (F.M.1), during weaning (F.M.3), and postweaning (F.M.6). To study the alterations in intestinal microflora, serum metabolites, and their correlation before and postweaning. We found that with the growth and weaning progress of donkey foals, the intestinal microbiota of donkey foals underwent obvious changes, and the diversity of fecal bacteria increased (Chao1 and Shannon indexes). The main intestinal microbial flora of donkey foals include Bacteroides and Firmicutes. We found many microbiota that are associated with immunity and digestion in the postweaning group, such as Verrucomicrobiales, Clostridia, Oscillospiraceae, Akkermansia, and Rikenellaceae, which can be considered microbial markers for the transition from liquid milk to solid pellet feed. Clostridia and Oscillospiraceae can produce organic acids, including butyric acid and acetic acid, which are crucial for regulating the intestinal microecological balance of donkeys. Furthermore, the metabolome showed that the serum metabolites enriched before and postweaning were mainly related to arachidonic acid metabolism and riboflavin metabolism. Riboflavin was associated with the development of the small intestine and affected the absorption of the small intestine. We also found that the changes in the gut microbiome of the foals were significantly correlated with changes in serum metabolites, including lysophosphatidylcholine (LPC; 12,0) and positively correlated with Lachnoclostridium and Roseburia. To summarize, this study provides theoretical data for the changes in the intestinal microbiome and serum metabolism during the entire weaning period of donkey foals.

Bikeshare, as a convenient transport mode, can address the first- and last-mile travel needs of metro trips while generating many environmental benefits, such as reducing the use of environmentally unfriendly transport modes and lowering the carbon emissions of the urban transportation system. This paper takes bikeshare as a feeder mode of metro stations (BS-FMMS) as the research object and compares the spatial and temporal differences in the carbon emission reduction benefits of BS-FMMS on workdays and non-workdays by using the framework of BS-FMMS carbon reduction benefit analysis and the methods of time-series analysis, spatial aggregation analysis, and box plot analysis. The results show that the carbon emission reduction benefit of bikeshare has obvious morning and evening peaks on workdays, while it tends to be stable without obvious peaks during the day on non-workdays. From the perspective of spatial distribution, the carbon emission reduction benefits of BS-FMMS are more significant in the metro station areas in the south of Baoan district, the west of Nanshan district, the central of Longhua district, and the south of Futian district in Shenzhen city, and the metro stations where the carbon emission reduction benefits of the non-workday are greater than those of the workday are mainly concentrated in Nanshan district, Futian district, and Luohu district. There is a significant positive correlation between BS-FMMS ridership and carbon emission reduction. These findings can provide clear policy implications for the decarbonization development of urban transportation systems.

ABSTRACT: Pigeons are considered less susceptible, and display few or no clinical signs to infection with avian influenza virus (AIV). Melanoma differentiation-associated gene 5 (MDA5), an important mediator in innate immunity, has been linked to the virus resistance. In this study, the pigeon MDA5 (piMDA5) was cloned. The bioinformatics analysis showed that the C-terminal domain (CTD) of MDA5 is highly conserved among species while the N-terminal caspase recruitment domain (CARD) is variable. Upon infection with Newcastle diseases virus (NDV) and AIV, piMDA5 was upregulated in both pigeons and pigeon embryonic fibroblasts (PEFs). Further study found that overexpression of piMDA5 mediated the activation of interferons (IFNs) and IFN-stimulated genes (ISGs) while inhibiting NDV replication. Conversely, the knockdown of piMDA5 promoted NDV replication. Additionally, CARD was found to be essential for the activation of IFN-β by piMDA5. Furthermore, pigeon MDA5, chicken MDA5, and human MDA5 differ in inhibiting viral replication and inducing ISGs expression. These findings suggest that MDA5 contributes to suppressing viral replication by activating the IFN signal pathway in pigeons. This study provides valuable insight into the role of MDA5 in pigeons and a better understanding of the conserved role of MDA5 in innate immunity during evolution.

Background: Atrial fibrillation is one of the most common cardiac arrhythmias. Myocardial fibrosis is closely associated with atrial remodeling, which leads to heightened risk of atrial fibrillation. This study aimed to explore whether forkhead box protein O3 (FOXO3a) impacts myocardial fibrosis incidence by regulating mitophagy. Methods: Cell viability was assessed by cell counting kit-8 (CCK-8) assays. The expression of vimentin and cytochrome C was detected by immunofluorescence assays. Quantitative real-time polymerase chain reaction (PCR) was used to analyze the relative mRNA level of FOXO3a. Expression of FOXO3a, phosphorylated FOXO3a, Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), matrix metalloprotease 9 (MMP9), light chain 3 (LC3), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), Parkin, and sequestosome-1 (p62) proteins were determined by western blotting. 5-ethynyl-2′-deoxyuridine (EDU) incorporation was employed to measure cell proliferation. Changes in mitochondrial membrane potential were determined by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining. A wound healing assay was used to examine cell migration, and the levels of reactive oxygen species were determined by flow cytometry. Results: The expression of FOXO3a was upregulated in cardiac fibroblasts treated with angiotensin II (AngII), while the expression of phosphorylated FOXO3a was downregulated under these conditions. FOXO3a knockdown significantly inhibited the proliferation, migration and collagen secretion of cardiac fibroblasts treated with AngII. The ratio of LC3 II/I as well as expression of PINK1 and Parkin was increased, and the expression of p62 was decreased, in cardiac fibroblasts treated with AngII. Moreover, these effects were limited by FOXO3a knockdown. Finally, the mitophagy inducer everolimus (RAD001) attenuated the suppressive effect of FOXO3a knockdown on cardiac fibroblast activation. Conclusions: FOXO3a promotes the progress of myocardial fibrosis by triggering mitophagy in cardiac fibroblasts.

The ability of stimulator of interferon genes (STING) to activate interferon (IFN) responses during RNA virus infection has been demonstrated in different mammalian cells. Despite being the host of numerous RNA viruses, the role of STING in bats during RNA virus infection has not been elucidated. In this study, we identified and cloned the STING gene of the Brazilian free-tailed bat Tadarida brasiliensis (T. brasiliensis) and tested its ability to induce IFN-β by overexpressing and knocking down bat STING (BatSTING) in T. brasiliensis 1 lung (TB1 Lu) cells. In addition, we used green fluorescent protein (GFP)-labeled vesicular stomatitis virus (VSV) VSV-GFP as a model to detect the antiviral activity of BatSTING. The results showed that overexpression of STING in TB1 Lu cells stimulated by cGAS significantly inhibited RNA virus replication, and the antiviral activities were associated with its ability to regulate basal expression of IFN-β and some IFN stimulated genes (ISGs). We also found that BatSTING was able to be activated after stimulation by diverse RNA viruses. The results of TB1 Lu cells with STING deficiency showed that knockdown of BatSTING severely hindered the IFN-β response triggered by VSV-GFP. Based on this, we confirm that BatSTING is required to induce IFN-β expression during RNA virus infection. In conclusion, our experimental data clearly show that STING in bat hosts plays an irreplaceable role in mediating IFN-β responses and anti-RNA virus infection.

Abstract Innate immunity plays an essential role in preventing the invasion of pathogenic microorganisms. However, innate immunity is a double-edged sword, whose excessive activation is detrimental to immune homeostasis and even leads to a “cytokine storm” of the infected host. The host develops a series of negative regulatory mechanisms to balance the immune response. Here, we report a negative regulatory mechanism of chicken innate immunity mediated by miRNA. In the GEO database, we found that miR-126-5p was markedly up-regulated in chickens infected by RNA viruses. Upregulation of miR-126-5p by RNA virus was then further shown via both a cell model and in vivo tests. Overexpression of miR-126-5p significantly inhibited the expression of interferon and inflammatory cytokine-related genes induced by RNA viruses. The opposite result was achieved after the knockdown of miR-126-5p expression. Bioinformatics analysis identified TRAF3 as candidate target gene of miR-126-5p. Experimentally, miR-126-5p can target TRAF3, as shown by the effects of miR-126-5p on the endogenous expression of TRAF3, and by the TRAF3 3'UTR driven luciferase reporter assay. Furthermore, we demonstrated that miR-126-5p negatively regulated innate immunity by blocking the MAVS-TRAF3-TBK1 axis, with a co-expression assay. Overall, our results suggest that miR-126-5p is involved in the negative regulation of chicken innate immunity, which might contribute to maintaining immune balance.

In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.

Species within the genus Equus are valued for their draft ability. Skeletal muscle forms the foundation of the draft ability of Equus species; however, skeletal muscle development-related conserved genes and their target miRNAs are rarely reported for Equus. In this study, a comparative genomics analysis was performed among five species (horse, donkey, zebra, cattle, and goat), and the results showed that a total of 15,262 (47.43%) genes formed the core gene set of the five species. Only nine chromosomes (Chr01, Chr02, Chr03, Chr06, Chr10, Chr18, Chr22, Chr27, Chr29, and Chr30) exhibited a good collinearity relationship among Equus species. The micro-synteny analysis results showed that TPM3 was evolutionarily conserved in chromosome 1 in Equus. Furthermore, donkeys were used as the model species for Equus to investigate the genetic role of TPM3 in muscle development. Interestingly, the results of comparative transcriptomics showed that the TPM3 gene was differentially expressed in donkey skeletal muscle S1 (2 months old) and S2 (24 months old), as verified via RT-PCR. Dual-luciferase test analysis showed that the TPM3 gene was targeted by differentially expressed miRNA (eca-miR-1). Furthermore, a total of 17 TPM3 gene family members were identified in the whole genome of donkey, and a heatmap analysis showed that EaTPM3-5 was a key member of the TPM3 gene family, which is involved in skeletal muscle development. In conclusion, the TPM3 gene was conserved in Equus, and EaTPM3-5 was targeted by eca-miR-1, which is involved in skeletal muscle development in donkeys.

Abstract Interferon regulatory factors (IRFs) play a key role in many aspects of immune response, and IRF1, IRF3, and IRF7 are positive regulators of IFN induction in mammals. However, IRF3, as the most critical regulatory factor in mammals, is naturally absent in birds, which attracts us to study the functions of other members of the avian IRF family. In the present study, we cloned goose IRF1 (GoIRF1) and conducted a series of bioinformatics analyses to compare the protein homology of GoIRF1 with that of IRF1 in other species. The overexpression of GoIRF1 in DF-1 cells induced the activation of IFN-β, and this activation is independent of the dosage of the transfected GoIRF1 plasmids. The overexpression of GoIRF1 in goose embryonic fibroblasts (GEFs) induced the expression of IFNs, proinflammatory cytokines, and IFN-stimulated genes (ISGs); it also inhibited the replication of green fluorescent protein (GFP)-tagged Newcastle disease virus (NDV) (NDV-GFP) and GFP-tagged vesicular stomatitis virus (VSV) (VSV-GFP). Our results suggest that GoIRF1 is an important regulator of IFNs, proinflammatory cytokines, and ISGs and plays a role in antiviral innate immunity in geese.

Compatibility between asphalt and polyurethane (PU) additives is the most challenging issue affecting the performance of PU modified asphalt. This study aims to investigate the influence of shearing temperature and PU content on compatibility between PU and asphalt via experiments and molecular dynamics (MD) simulation. Herein, the molecular models of base asphalt, PU molecules, and PU/asphalt blends were first constructed in Materials Studio (MS) software. Based on a reasonable model, the influence of shearing temperature on the storage stability of PU modified asphalt was measured by calculating the solubility parameters (δ) and diffusion coefficient. In addition, the effect of PU content on the compatibility of PU and asphalt was evaluated through cohesive energy density (CED), hydrogen bonding, and radial distribution function (RDF). Finally, the simulation results were compared with results from the segregation test and fluorescence microscope (FM). The simulation results indicate that PU and asphalt obtain optimal compatibility when the temperature is 135 °C, at which the diffusion coefficient of PU modified asphalt is enhanced significantly. The CED of modified asphalt is enhanced with the increase of PU content. Meanwhile, PU shows a significant effect on the RDF of each asphalt component. All these changes in asphalt molecules demonstrate that PU plays a positive role in improving the tightness of asphalt molecular structure, and the optimal content is 15 wt%. The simulation results are consistent with experimental results.

ABSTRACT: The mammalian-derived MDCK cells are the most widely used for avian virus vaccine production at present. The use of heterologous cell systems for avian virus preparation may cause security risks. An avian cell line is available for avian virus vaccines urgently needed. In this study, a peacock immortalized fibroblast cell line that is suitable for avian virus vaccine production was generated. The primary peacock fibroblast cells were prepared, and the immortal cells PEF-1 were obtained by transferring hTERT into the primary cells and screening with G418. The PEF-1 has high cell viability and expresses exogenous TERT protein. More importantly, the virus replication ability was stronger in PEF-1 than in MDCK cells as evaluated by virus fluorescence and TCID50, after being infected with NDV-GFP, VSV-GFP, and AIV. In conclusion, the peacock immortalized PEF cells are expected to be used for the production of peacock and other avian virus vaccines.

China’s energy consumption and its growth trend determine the domestic energy production and supply pattern. The understanding of energy consumption and its changes will help to enhance urban resilience. Based on the improved input-output model and hypothesis extraction model, this paper aims at constructing the energy-industry connection model, analyzing the energy transfer implied in the economic activities of various industrial sectors, and examining the energy transfer effect between the supply-side (SS) and demand-side industry sectors of the Shaanxi Province. The results showed that, in 2017, the energy industrial sector was the most energy consumption industry in Shaanxi Province. The industrial energy sector belonged to the net energy output industrial sector, and the energy products were transferred to other industrial sectors. This paper can provide a scientific basis for the energy SS reform, adjusting the industrial layout of regions in the Yellow River Basin.

Stimulator of the interferon gene (STING) is involved in mammalian antiviral innate immunity as an interferon (IFN) activator. However, there is still a lack of clarity regarding the molecular characterization of goose STING (GoSTING) and its role in the innate immune response. In the present study, we cloned GoSTING and performed a series of bioinformatics analyses. GoSTING was grouped into avian clades and showed the highest sequence similarity to duck STING. The in vitro experiments showed that the mRNA levels of GoSTING, IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines were significantly upregulated in goose embryo fibroblast cells (GEFs) infected with Newcastle disease virus (NDV). Overexpression of GoSTING in DF-1 cells and GEFs strongly activated the IFN-β promoter as detected by a dual-luciferase reporter assay. Furthermore, overexpression of GoSTING induced the expression of other types of IFN, ISGs, and proinflammatory cytokines and inhibited green fluorescent protein (GFP)-tagged NDV (NDV-GFP) and GFP-tagged vesicular stomatitis virus (VSV) (VSV-GFP) replication in vitro. In conclusion, these data suggest that GoSTING is an important regulator of the type I IFN pathway and is critical in geese’s innate immune host defense against RNA viruses.

Streptococcus suis is a significant zoonotic pathogen that is a great threat not only to the swine industry but also to human health, causing arthritis, meningitis, and even streptococcal toxic shock-like syndrome. Owing to its many serotypes and high geographic variability, an efficacious cross-protective S. suis vaccine is not readily available. Therefore, this study aimed to design a universal multi-epitope vaccine (MVHP6) that involved three highly immunogenic proteins of S. suis, namely, the surface antigen containing a glycosaminoglycan binding domain (HP0197), endopeptidase (PepO), and 6-phosphogluconate dehydrogenase (6PGD). Forecasted T-cell and B-cell epitopes with high antigenic properties and a suitable adjuvant were linked to construct a multi-epitope vaccine. In silico analysis showed that the selected epitopes were conserved in highly susceptible serotypes for humans. Thereafter, we evaluated the different parameters of MVHP6 and showed that MVHP6 was highly antigenic, non-toxic, and non-allergenic. To verify whether the vaccine could display appropriate epitopes and maintain high stability, the MVHP6 tertiary structure was modeled, refined, and validated. Molecular docking studies revealed a strong binding interaction between the vaccine and the toll-like receptor (TLR4), whereas molecular dynamics simulations demonstrated the vaccine’s compatibility, binding stability, and structural compactness. Moreover, the in silico analysis showed that MVHP6 could evoke strong immune responses and enable worldwide population coverage. Moreover, MVHP6 was cloned into the pET28a (+) vector in silico to ensure the credibility, validation, and proper expression of the vaccine construct. The findings suggested that the proposed multi-epitope vaccine can provide cross-protection against S. suis infections.

Bats are important hosts for various zoonotic viral diseases. However, they rarely show signs of disease infection with such viruses. As the first line for virus control, the innate immune system of bats attracted our full attention. In this study, the Tadarida brasiliensis MDA5 gene (batMDA5), a major sensor for anti-RNA viral infection, was first cloned, and its biological functions in antiviral innate immunity were identified. Bioinformatics analysis shows that the amino acid sequence of batMDA5 is poorly conserved among species, and it is evolutionarily closer to humans. The mRNA of batMDA5 was significantly upregulated in Newcastle disease virus (NDV), avian influenza virus (AIV), and vesicular stomatitis virus (VSV)-infected bat TB 1 Lu cells. Overexpression of batMDA5 could activate IFNβ and inhibit vesicular stomatitis virus (VSV-GFP) replication in TB 1 Lu cells, while knockdown of batMDA5 yielded the opposite result. In addition, we found that the CARD domain was essential for MDA5 to activate IFNβ by constructing MDA5 domain mutant plasmids. These results indicated that bat employs a conserved MDA5 gene to trigger anti-RNA virus innate immune response. This study helps understand the biological role of MDA5 in innate immunity during evolution.

S. suis is an important zoonotic pathogen from sick and recessive carrier pigs that poses a serious threat to animal husbandry production and public health. It usually causes horizontal transmission among pigs. The morbidity and mortality of this disease are very high. Human infection is caused through direct or indirect contact with sick pigs. The two large-scale outbreaks in China were due to the outbreak of S. suis on pig farms, which spread to human infection; thus, detecting S. suis in pig herds is crucial. At present, the commercial S. suis ELISA type 2 kits on the market can only detect single serotypes, high probabilities of interaction reactions, and biosafety risks when using inactivated S. suis as an antigen. Phosphate-3-glyceraldehyde dehydrogenase (GAPDH), muramidase-released protein (MRP), and dihydrolipoamide dehydrogenase (DLDH) are important S. suis type 2, S. suis type 7, and S. suis type 9 protective antigens. This study purified the GMD protein (B-cell-dominant epitopes of GAPDH, MRP, and DLDH antigens) and used a diverse combination of dominant epitopes of the multiple different antigens as coated antigens, improving the sensitivity and safety of the indirect ELISA experiments. An indirect ELISA method (GMD-ELISA) was developed for detecting S. suis antibodies. The antigen—antibody response was optimized using checkerboard titration. The results of testing using ELISA for Salmonella enterica (S. enterica), Escherichia coli (E. coli), Staphylococcus aureus (SA), and Streptococcus pyogenes (S. pyogenes) were all negative, indicating that this method had strong specificity. The results were still positive when the dilution ratio of S. suis-positive serum reached 1:6, 400, thus indicating that the method had high sensitivity. The results of the reproducibility assay for indirect ELISA showed that the intra-assay coefficient of variation and the inter-assay coefficient of variation were less than 10%, indicating that the method had good repeatability. We investigated the seroprevalence of S. suis in 167 serum samples collected in East China, and 33.5% of the samples were positive for antibodies against S. suis, indicating that the prevalence of S. suis is high in pig farms in Eastern China. The novel GMD-ELISA is a convenient, sensitive, and specific diagnostic method that provides technical support for rapid diagnosis and epidemiological investigation.

Abstract Non-structural protein 1 (NS1) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. In this study, an acetylation modification was identified at the K108 residue of the NS1 protein of H1N1 influenza virus. To further explore the function of the K108 acetylation modification of the NS1 protein, a deacetylation-mimic mutation (K108R) and a constant acetylation-mimic mutation (K108Q) were introduced into the NS1 protein in the background of A/WSN/1933 H1N1 (WSN), resulting in two mutant viruses (WSN-NS1-108R and WSN-NS1-108Q). In vitro and mouse studies showed that the deacetylation-mimic mutation K108R in the NS1 protein attenuated the replication and virulence of WSN-NS1-108R, while the constant acetylation-mimic mutant virus WSN-NS1-108Q showed similar replication and pathogenicity as the wild-type WSN virus (WSN-wt). The results indicated that acetylation at K108 of the NS1 protein has an important role in the replication and virulence of influenza virus. To further explore the potential mechanism, the type I interferon (IFN-I) antagonistic activity of the three NS1 proteins (NS1-108Q, NS1-108R, and NS1-wt) was compared in cells, which showed that the K108R mutation significantly attenuated the IFN-β antagonistic activity of the NS1 protein compared with NS1-wt and NS1-108Q. Both NS1-wt and NS1-108Q inhibited the IFN-β response activated by RIG-I CARD domain, MAVS, TBK1, and IRF3 more efficiently than the NS1-108R protein in cells. Taken together, the results indicated that acetylation at NS1 K108 is important for the IFN antagonistic activity of the NS1 protein and virulence of the influenza virus.

Chickens are the natural host of Newcastle disease virus (NDV) and avian influenza virus (AIV). The discovery that the RIG-I gene, the primary RNA virus pattern recognition receptor (PRR) in mammals, is naturally absent in chickens has directed attention to studies of chicken RNA PRRs and their functions in antiviral immune responses. Here, we identified Asp-Glu-Ala-Asp (DEAD)-box helicase 1 (DDX1) as an essential RNA virus PRR in chickens and investigated its functions in anti-RNA viral infections. The chDDX1 gene was cloned, and cross-species sequence alignment and phylogenetic tree analyses revealed high conservation of DDX1 among vertebrates. A quantitative RT-PCR showed that chDDX1 mRNA are widely expressed in different tissues in healthy chickens. In addition, chDDX1 was significantly upregulated after infection with AIV, NDV, or GFP-expressing vesicular stomatitis virus (VSV-GFP). Overexpression of chDDX1 in DF-1 cells induced the expression of IFN-β, IFN-stimulated genes (ISGs), and proinflammatory cytokines; it also inhibited NDV and VSV replications. The knockdown of chDDX1 increased the viral yield of NDV and VSV and decreased the production of IFN-β, which was induced by RNA analog polyinosinic-polycytidylic acid (poly[I:C]), by AIV, and by NDV. We used a chicken IRF7 (chIRF7) knockout DF-1 cell line in a series of experiments to demonstrate that chDDX1 activates IFN signaling via the chIRF7 pathway. Finally, an in-vitro pulldown assay showed a strong and direct interaction between poly(I:C) and the chDDX1 protein, indicating that chDDX1 may act as an RNA PRR during IFN activation. In brief, our results suggest that chDDX1 is an important mediator of IFN-β and is involved in RNA- and RNA virus-mediated chDDX1-IRF7-IFN-β signaling pathways.

Pseudomonas aeruginosa is a Gram-negative pathogen that causes a variety of infections in humans and animals. Due to the inappropriate use of antibiotics, multi-drug resistant (MDR) P. aeruginosa strains have emerged and are prevailing. In recent years, cow mastitis caused by MDR P. aeruginosa has attracted attention. In this study, a microbial community analysis revealed that P. aeruginosa could be a cause of pathogen-induced cow mastitis. Five MDR P. aeruginosa strains were isolated from milk diagnosed as mastitis positive. To seek an alternative antibacterial agent against MDR, P. aeruginosa, a lytic phage, designated vB_PaeS_PAJD-1 (PAJD-1), was isolated from dairy farm sewage. PAJD-1 was morphologically classified as Siphoviridae and was estimated to be about 57.9 kb. Phage PAJD-1 showed broad host ranges and a strong lytic ability. A one-step growth curve analysis showed a relatively short latency period (20 min) and a relatively high burst size (223 PFU per infected cell). Phage PAJD-1 remained stable over wide temperature and pH ranges. Intramammary-administered PAJD-1 reduced bacterial concentrations and repaired mammary glands in mice with mastitis induced by MDR P. aeruginosa. Furthermore, the cell wall hydrolase (termed endolysin) from phage PAJD-1 exhibited a strong bacteriolytic and a wide antibacterial spectrum against MDR P. aeruginosa. These findings present phage PAJD-1 as a candidate for phagotherapy against MDR P. aeruginosa infection.

Mastitis caused by Escherichia coli (E. coli) remains a threat to dairy animals and impacts animal welfare and causes great economic loss. Furthermore, antibiotic resistance and the lagged development of novel antibacterial drugs greatly challenge the livestock industry. Phage therapy has regained attention. In this study, three lytic phages, termed vB_EcoM_SYGD1 (SYGD1), vB_EcoP_SYGE1 (SYGE1), and vB_EcoM_SYGMH1 (SYGMH1), were isolated from sewage of dairy farm. The three phages showed a broad host range and high bacteriolytic efficiency against E. coli from different sources. Genome sequence and transmission electron microscope analysis revealed that SYGD1 and SYGMH1 belong to the Myoviridae, and SYGE1 belong to the Autographiviridae of the order Caudovirales. All three phages remained stable under a wide range of temperatures or pH and were almost unaffected in chloroform. Specially, a mastitis infected cow model, which challenged by a drug resistant E. coli, was used to evaluate the efficacy of phages. The results showed that the cocktails consists of three phages significantly reduced the number of bacteria, somatic cells, and inflammatory factors, alleviated the symptoms of mastitis in cattle, and achieved the same effect as antibiotic treatment. Overall, our study demonstrated that phage cocktail may be a promising alternative therapy against mastitis caused by drug resistant E. coli.

Remote sensing technology is important for soil organic matter (SOM) estimation, but existing studies have mainly relied on a single data source. This limitation makes it difficult to simultaneously ensure high spatial resolution, high spectral accuracy and refined temporal granularity simultaneously, which cannot meet the requirements of the spatiotemporal dynamics representation. This study aimed to introduce a new remote sensing image source into SOM modeling and spatiotemporal estimation generated by fusing together Sentinel-2 and Sentinel-3 remote sensing images that have a 5-day revisit cycle; 10 m spatial resolution; and 21 different bands in blue, green, red and NIR spectral ranges. According to the image fusion process, a total of 52 available images were acquired between November 2016 and December 2018 in Donghai County, China. The fused images were used for SOM estimation model associated with 107 field samples. The results indicated that, first, the optimal model consisted of the band reflectivity (B20) and RVI (B18/B9), which were derived from the fused images, and the R2 approached 0.7 in the two phases of the synchronized data. Second, the modeling accuracy was influenced to some extent by the actual SOM content. The R2 values exceeded 0.75 when the SOM content was higher than 24 g/kg, while the R2 was even lower than 0.35 when the SOM content was lower. Third, the averaged SOM contents remained stable in general, while the seasonal variances can also be found during the two-year interval. The SOM contents maintained a low level during autumn and winter, while higher SOM levels were found in the spring and summer. Finally, the spatial variations could be described as ‘low in the west and high in the east’. In summary, the spatiotemporal dynamics of SOM highlighted the necessity of modeling with fused remote sensing images, and more effective modeling could be expected with the continued increase in SOM in future.

Reassortant variant viruses generated between 2009 H1N1 pandemic influenza virus [A(H1N1)pdm09] and endemic swine influenza viruses posed a potential risk to humans. Surprisingly, genetic analysis showed that almost all of these variant viruses contained the M segment from A(H1N1)pdm09, which originated from Eurasian avian-like swine influenza viruses. Studies have shown that the A(H1N1)pdm09 M gene is critical for the transmissibility and pathogenicity of the variant viruses. However, the M gene encodes two proteins, M1 and M2, and which of those plays a more important role in virus pathogenicity remains unknown. In this study, the M1 and M2 genes of A(H1N1)pdm09 were replaced with those of endemic H3N2 swine influenza virus, respectively. The chimeric viruses were rescued and evaluated in vitro and in mice. Both M1 and M2 of H3N2 affected the virus replication in vitro. In mice, the introduction of H3N2 M1 attenuated the chimeric virus, where all the mice survived from the infection, compared with the wild type virus that caused 100 % mortality. However, the chimeric virus containing H3N2 M2 was still virulent to mice, and caused 16.6% mortality, as well as similar body weight loss to the wild type virus infected group. Compared with the wild type virus, the chimeric virus containing H3N2 M1 induced lower levels of inflammatory cytokines and higher levels of anti-inflammatory cytokines, whereas the chimeric virus containing H3N2 M2 induced substantial pro-inflammatory responses, but higher levels of anti-inflammatory cytokines. The study demonstrated that Eurasian avian-like M1 played a more important role than M2 in the pathogenicity of A(H1N1)pdm09 in mice.

The stimulator of interferon genes (STING) protein has been shown to play a pivotal role in response to both cytosolic RNA and dsDNA to elicit interferon (IFN) production in mammals. However, the role of duck STING (DuSTING) in antiviral innate immunity, especially in anti-RNA virus infection, has yet to be elucidated. In this study, the function of DuSTING in IFN induction and its role in anti-RNA virus infections were studied. DuSTING was amplified via reverse transcription-polymerase chain reaction (RT-PCR) from Pekin duck, showing that its cDNA sequence contains an open reading frame (ORF) of 1,149 bp and encodes 382 amino acids (aa). Sequence alignment showed that DuSTING protein shares 71.1, 43.4, and 33.3% identity with chickens, humans, and zebra fish, respectively. Overexpression of DuSTING in duck embryo fibroblasts (DEFs) strongly activated IFN-β promotor activity. Deletion mutant analysis revealed that the first 42 aa containing the first transmembrane (TM) domains and the last 32 aa containing a part of the C-terminal tail (CTT) are essential for its IFN-β activation. In vitro experiments showed that the mRNA levels of DuSTING and IFNs were all upregulated when the DEFs were infected with H9N2 avian influenza virus (AIV) SH010, while overexpression of DuSTING inhibited the replication of this virus. In vivo studies showed that DuSTING mRNA was widely expressed in different tissues, and was up-regulated in the spleen and lung of ducks challenged with SH010. In conclusion, our results indicate that DuSTING is an essential IFN mediator and plays a role in anti-RNA virus innate immunity.

Streptococcus suis (S. suis) serotype 2 (SS2) is the causative agent of swine streptococcosis and can cause severe diseases in both pigs and humans. Although the traditional inactive vaccine can protect pigs from SS2 infection, novel vaccine candidates are needed to overcome its shortcomings. Three infection-associated proteins in S. suis—muramidase-released protein (MRP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and DLD, a novel putative dihydrolipoamide dehydrogenase—have been previously identified by immunoproteomic assays. In this study, the effective immune protection of the recombinant trivalent protein GAPDH-MRP-DLD (JointS) against SS2, SS7, and SS9 was determined in zebrafish. To improve the immune efficacy of JointS, monophosphoryl lipid A (MPLA) as a TLR4 agonist adjuvant, which induces a strong innate immune response in the immune cells of mice and pigs, was combined with JointS to immunize the mice. The results showed that immunized mice could induce the production of a high titer of anti-S. suis antibodies; as a result, 100% of mice survived after SS2 infection. Furthermore, JointS provides good protection against virulent SS2 strain infections in piglets. Given the above, there is potential to develop JointS as a novel subunit vaccine for piglets to prevent infection by SS2 and other S. suis serotypes.

The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N5, N10-methylene tetrahydrofolate (N5, N10-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N5, N10-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.

Streptococcus suis (S. suis) is a gram-positive bacterium and zoonotic pathogen. Currently it poses a serious problem in the swine industry due to the emergence of antibiotic-resistant bacteria. Thus, novel antimicrobials against S. suis infections are urgently needed. In the previous study, a cell wall hydrolase or lysin derived from Streptococcus prophage phi5218, termed Ply5218, was identified. This lysin showed strong bacteriolytic activity against S. suis. In the current study, the in vitro data showed that after incubation with pig serum, the bacteriolytic efficacy of Ply5218 declined in a time-dependent manner. The in vivo assays indicated that a Ply5218 triple treatment (6, 24, and 48 h post infection) was effective against various serotypes of S. suis in a murine infection model. This regimen also alleviated streptococcal-induced clinical symptoms in piglets and significantly reduced the bacterial burden and levels of interleukin 6, a proinflammatory cytokine. This study indicates that Ply5218 shows strong antibacterial activity in pigs and has the potential to be used as a treatment for infectious diseases caused by S. suis.

The ability of stimulator of interferon genes (STING) to activate interferon (IFN) responses during RNA virus infection has been demonstrated in different mammalian cells. Despite being the host of numerous RNA viruses, the role of STING in bats during RNA virus infection has not been elucidated. In this study, we identified and cloned the STING gene of the Brazilian free-tailed bat Tadarida brasiliensis (T. brasiliensis) and tested its ability to induce IFN-ß by overexpressing and knocking down bat STING (BatSTING) in T. brasiliensis 1 lung (TB1 Lu) cells. In addition, we used green fluorescent protein (GFP)-labeled vesicular stomatitis virus (VSV) VSV-GFP as a model to detect the antiviral activity of BatSTING. The results showed that overexpression of STING in TB1 Lu cells stimulated by cGAS significantly inhibited RNA virus replication, and the antiviral activities were associated with its ability to regulate basal expression of IFN-ß and some IFN stimulated genes (ISGs). We also found that BatSTING was able to be activated after stimulation by diverse RNA viruses. The results of TB1 Lu cells with STING deficiency showed that knockdown of BatSTING severely hindered the IFN-ß response triggered by VSV-GFP. Based on this, we confirm that BatSTING is required to induce IFN-ß expression during RNA virus infection. In conclusion, our experimental data clearly show that STING in bat hosts plays an irreplaceable role in mediating IFN-ß responses and anti-RNA virus infection. [ABSTRACT FROM AUTHOR]