African swine fever virus pCP312R interacts with host RPS27A to shut off host protein translation and promotes viral replication.

African swine fever virus (ASFV) severely threatens the global economy and food security. ASFV encodes >150 genes, but the functions of most of them have yet to be characterized in detail. Here we explored the function of the ASFV CP312R gene and found that CP312R plays an essential role in ASFV replication. Knockout of the CP312R gene terminated viral replication and CP312R knockdown substantially suppressed ASFV infection in vitro. Furthermore, we resolved the crystal structure of pCP312R to 2.3 Å resolution and found that pCP312R has the potential to bind nucleic acids. LC-MS analysis and co-immunoprecipitation assay revealed that pCP312R interacts with RPS27A, a component of the 40S ribosomal subunit. Confocal microscopy showed the interaction between pCP312R and RPS27A leaded to a modification in the subcellular localization of this host protein, which suppresses host protein translation. Renilla-Glo luciferase assay and Ribopuromycylation analysis evidenced that knockout of RPS27A completely aborted the shutoff activity of pCP312R, and trans-complementation of RPS27A recovered pCP312R shutoff activity in RPS27A-knockout cells. Our findings shed light on the function of ASFV CP312R gene in virus infection, which triggers inhibition of host protein synthesis.

A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes.

African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.

A novel bacterium-like particles platform displaying antigens by new anchoring proteins induces efficacious immune responses.

Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.

Molecular Mechanisms of Intestinal Protection by Levilactobacillus brevis 23017 against Salmonella typhimurium C7731-Induced Damage: Role of Nrf2.

The treatment and prevention of pathogenic diseases by lactic acid bacteria (LAB) has attracted more and more attention. As a special LAB, Levilactobacillus brevis (L. brevis) has relatively less research on its antibacterial infection in vivo, and its protective effect and mechanism still need to be fully studied. In this study, we selected L. brevis 23017, which can regulate the intestinal immunity of the host animal and resist pathogen infection, to evaluate its protective role and potential molecular mechanisms in the mouse model of S. typhimurium C7731 infection. As expected, we confirmed that L. brevis 23017 reduced the diarrhea rate and increased the daily weight gain and survival rate of the mouse model, and inhibited S. typhimurium colonization in the jejunum and liver. It also reduced the level of oxidative damage and protected the integrity of intestinal tissue by increasing the activity of intestinal antioxidant enzymes (SOD, GSH-Px and T-AOC). From the perspective of intestinal mucosal barrier injury and repair, it was confirmed that L. brevis 23017 could increase the expression levels of intestinal tight junction proteins (ZO-1 and OCLN). Our research results also show that L. brevis 23017 inhibits the inflammatory response and promotes the occurrence of cellular immunity in the body by promoting the increase in IL-10 and inhibiting IL-13 in serum and intestinal tissue. Notably, L. brevis 23017 increased total secretory immunoglobulin A (SIgA) levels in the intestine, which were closely associated with elevated levels of IL-5, IL-13, pIgR, j-chain, and IgAα-chain. In addition, L. brevis 23017 increased the expression of antioxidant proteins Nrf2, NQO1, and HO-1 associated with Nrf2 signaling to inhibit intestinal oxidative damage. This mechanism may be responsible for its protective effect against S. typhimurium-infected intestine. Our study provides new evidence and theoretical support for the analysis of the anti-bacterial infection effect and mechanism of L. brevis, which will contribute to the development of L. brevis and the treatment of pathogenic bacteria intestinal infection.

Comprehensive Analysis of the Immunosuppressive Function of Regulatory T Cells in Human Hepatocellular Carcinoma Tissues.

BACKGROUND: Immune-based therapies are commonly employed to combat hepatocellular carcinoma (HCC). However, the presence of immune-regulating elements, especially regulatory T cells (Tregs), can dramatically impact the treatment efficacy. A deeper examination of the immune-regulation mechanisms linked to these inhibitory factors and their impact on HCC patient outcomes is warranted. METHODS: We employed multicolor fluorescence immunohistochemistry (mIHC) to stain Foxp3, cytokeratin, and nuclei on an HCC tissue microarray (TMA). Leveraging liver cancer transcriptome data from TCGA, we built a prognostic model focused on Treg-associated gene sets and represented it with a nomogram. We then sourced liver cancer single-cell RNA sequencing data (GSE140228) from the GEO database, selectively focusing on Treg subsets, and conducted further analyses, including cell-to-cell communication and pseudo-time trajectory examination. RESULTS: Our mIHC results revealed a more substantial presence of Foxp3+Tregs in HCC samples than in adjacent normal tissue samples (P < .001). An increased presence of Foxp3+Tregs in HCC samples correlated with unfavorable patient outcomes (HR = 1.722, 95% CI:1.023-2.899, P = .041). The multi-factorial prognosis model we built from TCGA liver cancer data highlighted Tregs as a standalone risk determinant for predicting outcomes (HR = 3.84, 95% CI:2.52-5.83, P < .001). Re-analyzing the scRNA-seq dataset (GSE140228) showcased distinctive gene expression patterns in Tregs from varying tissues. Interactions between Tregs and other CD4+T cell types were predominantly governed by the CXCL13/CXCR3 signaling pathway. Communication pathways between Tregs and macrophages primarily involved MIF-CD74/CXCR4, LGALS9/CD45, and PTPRC/MRC1. Additionally, macrophages could influence Tregs via HLA-class II and CD4 interactions. CONCLUSION: An elevated presence of Tregs in HCC samples correlated with negative patient outcomes. Elucidating the interplay between Tregs and other immune cells in HCC could provide insights into the modulatory role of Tregs within HCC tissues.

Rosemary leaf powder improves egg quality, antioxidant status, gut barrier function, and cecal microbiota and metabolites of late-phase laying hens.

This study sought to determine the effects of rosemary leaf powder (RP) on laying performance, egg quality, serum indices, gut barrier function, and cecal microbiota and metabolites of late-phase laying hens. A total of 84 "Jing Tint 6" laying hens at 65-week old were randomly divided into 2 groups and fed either a basal diet (CON) or a basal diet supplemented with 0.3% RP. Our study revealed that RP improved the Haugh unit and decreased yolk n-6/n-3 polyunsaturated fatty acid (PUFA) ratio of laying hens, increased serum superoxide dismutase (SOD), jejunal activities of SOD and catalase (CAT), and jejunal zonula occludens-1 (ZO-1) expression, as well as decreased serum tumor necrosis factor-α (TNF-α) level and jejunal TNF-α mRNA expression. Rosemary leaf powder markedly enhanced (P < 0.05) cecal abundances of Rikenellaceae, Rikenellaceae_RC9_gut_group, and Turicibacter, tended to promote (P = 0.076) butyrate concentration, and reduced (P < 0.05) cecal abundances of Erysipelatoclostridiaceae, Sutterellaceae, Fusobacteriaceae, Campylobacteraceae, Sutterella, Campylobacter, and Fusobacterium, which were closely linked with Haugh unit, yolk n-6/n-3 PUFA ratio, serum SOD and TNF-α. In addition, RP altered the metabolic functions of cecal microbiota and enhanced the abundances of butyrate-synthesizing enzymes, including lysine 2,3-aminomutase, ß-lysine 5,6-aminomutase, and 3-oxoacid CoA-transferase. Together, 0.3% RP has the potential to enhance egg quality by partially modulating serum antioxidant status, jejunal barrier function, and cecal microbiota structure and metabolites, indicating that RP could be considered a promising feed additive to promote the production performance of late-phase laying hens.

Transcriptional and ultrastructural changes of macrophages after african swine fever virus infection.

African swine fever (ASF) is a highly impactful infectious disease in the swine industry, leading to substantial economic losses globally. The causative agent, African swine fever virus (ASFV), possesses intricate pathogenesis, warranting further exploration. In this study, we investigated the impact of ASFV infection on host gene transcription and organelle changes through macrophage transcriptome sequencing and ultrastructural transmission electron microscopy observation. According to the results of the transcriptome sequencing, ASFV infection led to significant alterations in the gene expression pattern of porcine bone marrow derived macrophages (BMDMs), with 2404 genes showing upregulation and 1579 genes downregulation. Cytokines, and chemokines were significant changes in the expression of BMDMs; there was significant activation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors. According to the observation of the ultrastructure, mitochondrial damage and mitochondrial autophagy were widely present in ASFV-infected cells. The reduced number of macrophage pseudopodia suggested that virus-induced structural changes may compromise pathogen recognition, phagocytosis, and signal communication in macrophages. Additionally, the decreased size and inhibited acidification of secondary lysosomes in macrophages implied suppressed phagocytosis. Overall, ASFV infection resulted in significant changes in the expression of cytokines and chemokines, accompanied by the activation of NLR and TLR signaling pathways. We reported for the first time that ASFV infection led to a reduction in pseudopodia numbers and a decrease in the size and acidification of secondary lysosomes.

Lactobacilli-derived adjuvants combined with immunoinformatics-driven multi-epitope antigens based approach protects against Clostridium perfringens in a mouse model.

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Proof of concept in utilizing the peptidoglycan skeleton of pathogenic bacteria as antigen delivery platform for enhanced immune response.

Subunit vaccines are becoming increasingly important because of their safety and effectiveness. However, subunit vaccines often exhibit limited immunogenicity, necessitating the use of suitable adjuvants to elicit robust immune responses. In this study, we demonstrated for the first time that pathogenic bacteria can be prepared into a purified peptidoglycan skeleton without nucleic acids and proteins, presenting bacterium-like particles (pBLP). Our results showed that the peptidoglycan skeletons screened from four pathogens could activate Toll-like receptor1/2 receptors better than bacterium-like particles from Lactococcus lactis in macrophages. We observed that pBLP was safe in mouse models of multiple ages. Furthermore, pBLP improved the performance of two commercial vaccines in vivo. We confirmed that pBLP successfully loaded antigens onto the surface and proved to be an effective antigen delivery platform with enhanced antibody titers, antibody avidity, balanced subclass distribution, and mucosal immunity. These results indicate that the peptidoglycan skeleton of pathogenic bacteria represents a new strategy for developing subunit vaccine delivery systems.

Precursor exhausted CD8+T cells in colorectal cancer tissues associated with patient's survival and immunotherapy responsiveness.

Exhausted CD8+T cells represent a distinct cellular lineage that emerges during both chronic infections and cancers. Recent studies have shown that persistent antigen exposure can drive the differentiation of precursor exhausted CD8+T cells, termed Tpex cells, which are characterized as TCF-1+PD-1+CD8+T cells. Elevated Tpex cell frequencies in the tumor microenvironment (TME) are associated with improved overall survival (OS) in cancer patients and heightened responsiveness to anti-PD-1 therapy. In our present study, we utilized multi-color immunohistochemistry (mIHC) to determine the localization and clinical implications of tumor-infiltrating Tpex cells within the TME of human colorectal cancer (CRC) tissues. We also conducted a multi-omics integrative analysis using single-cell RNA sequencing (scRNA-seq) data derived from both the murine MC38 tumor model and human CRC tissues. This analysis helped delineate the transcriptional and functional attributes of Tpex cells within the CRC TME. Furthermore, we employed spatial transcriptome sequencing data from CRC patients to investigate the interactions between Tpex cells and other immune cell subsets within the TME. In conclusion, our study not only established a method for Tpex cell detection using mIHC technology but also confirmed that assessing Tpex cells within the CRC TME could be indicative of patients' survival. We further uncovered the transcriptional and functional characteristics of Tpex cells in the TME and ascertained their pivotal role in the efficacy of immunotherapy against CRC.

Biogenic Selenium Nanoparticles Synthesized by L. brevis 23017 Enhance Aluminum Adjuvanticity and Make Up for its Disadvantage in Mice.

The most popular vaccine adjuvants are aluminum ones, which have significantly reduced the incidence and mortality of many diseases. However, aluminum-adjuvanted vaccines are constrained by their limited capacity to elicit cellular and mucosal immune responses, thus constraining their broader utilization. Biogenic selenium nanoparticles are a low-cost, environmentally friendly, low-toxicity, and highly bioactive form of selenium supplementation. Here, we purified selenium nanoparticles synthesized by Levilactobacillus brevis 23017 (L-SeNP) and characterized them using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, scanning electron microscopy, and transmission electron microscopy. The results indicate that the L-SeNP has a particle size ranging from 30 to 200 nm and is coated with proteins and polysaccharides. Subsequently, we assessed the immune-enhancing properties of L-SeNP in combination with an adjuvant-inactivated Clostridium perfringens type A vaccine using a mouse model. The findings demonstrate that L-SeNP can elevate the IgG and SIgA titers in immunized mice and modulate the Th1/Th2 immune response, thereby enhancing the protective effect of aluminum-adjuvanted vaccines. Furthermore, we observed that L-SeNP increases selenoprotein expression and regulates oxidative stress in immunized mice, which may be how L-SeNP regulates immunity. In conclusion, L-SeNP has the potential to augment the immune response of aluminum adjuvant vaccines and compensate for their limitations in eliciting Th1 and mucosal immune responses.

TMP269, a small molecule inhibitor of class IIa HDAC, suppresses RABV replication in vitro.

TMP269, a small molecular inhibitor of IIa histone deacetylase, plays a vital role in cancer therapeutic. However, the effect of TMP269 on the regulation of viral replication has not been studied. In the present study, we found that TMP269 treatment significantly inhibited RABV replication at concentrations without significant cytotoxicity in a dose-dependent manner. In addition, TMP269 can reduce the viral titers and protein levels of RABV at an early stage in the viral life cycle. RNA sequencing data revealed that immune-related pathways and autophagy-related genes were significantly downregulated after RABV infection treated with TMP269. Further exploration shows that autophagy enhances RABV replication in HEK-293T cells, while TMP269 can inhibit autophagy to decrease RABV replication. Together, these results provide a novel treatment strategy for rabies.

The Synergistic Activity of Rhamnolipid Combined with Linezolid against Linezolid-Resistant Enterococcus faecium.

Enterococci resistance is increasing sharply, which poses a serious threat to public health. Rhamnolipids are a kind of amphiphilic compound used for its bioactivities, while the combination of nontraditional drugs to restore linezolid activity is an attractive strategy to treat infections caused by these pathogens. This study aimed to investigate the activity of linezolid in combination with the rhamnolipids against Enterococcus faecium. Here, we determined that the rhamnolipids could enhance the efficacy of linezolid against enterococci infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, an anti-biofilm assay, molecular simulation dynamics, and mouse infection models. We identified that the combination of rhamnolipids and linezolid restored the linezolid sensitivity. Anti-biofilm experiments show that our new scheme can effectively inhibit biofilm generation. The mouse infection model demonstrated that the combination therapy significantly reduced the bacterial load in the feces, colons, and kidneys following subcutaneous administration. This study showed that rhamnolipids could play a synergistic role with linezolid against Enterococcus. Our combined agents could be appealing candidates for developing new combinatorial agents to restore antibiotic efficacy in the treatment of linezolid-resistant Enterococcus infections.

Effects of Catecholamine Stress Hormones Norepinephrine and Epinephrine on Growth, Antimicrobial Susceptibility, Biofilm Formation, and Gene Expressions of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a significant contributor to diarrhea. To determine whether ETEC-catecholamine hormone interactions contribute to the development of diarrhea, we tested the effects of catecholamine hormones acting on ETEC in vitro. The results showed that in the presence of norepinephrine (NE) and epinephrine (Epi), the growth of 9 out of 10 ETEC isolates was promoted, the MICs of more than 60% of the isolates to 6 antibiotics significantly increased, and the biofilm formation ability of 10 ETEC isolates was also promoted. In addition, NE and Epi also significantly upregulated the expression of the virulence genes feaG, estA, estB, and elt. Transcriptome analysis revealed that the expression of 290 genes was affected by NE. These data demonstrated that catecholamine hormones may augment the diarrhea caused by ETEC.

Whole-Genome Sequence and Pathogenicity Analysis of Providencia Heimbachae Causing Diarrhea in Weaned Piglets.

Providencia heimbachae was previously identified in piglets with post-weaned diarrhea and associated with hindlimb paralysis. However, the pathogenic mechanisms and virulence factors of P. heimbachae are not fully known. Whole-genome sequence analysis will be helpful to extend our understanding of the characterization of P. heimbachae at a genomic level. In this study, we sequenced the whole genome of P. heimbachae for the first time using PacBio RS II sequencers and assembled de novo through hierarchical genome assembly process (HGAP). Furthermore, we performed further genome annotation. The genome of P. heimbachae 99101 consists of a circular chromosome (4,262,828 bp) and a circular plasmid (231,957 bp) with G + C contents of 40.43 and 47.16%, respectively. Genome-wide sequence analysis yielded a total of 286 predicted virulence factors, 178 resistance genes, 17 chaperone protein manipulators of fimbriae, 47 genes involved in the encoding of flagellin, 12 cell membrane-associated virulence genes, 18 Enterobacteriaceae common antigens, etc. Based on genome analysis, we preliminarily confirmed through animal experiments that the capsule was the virulence factor of P. heimbachae causing hindlimb paralysis in animals. Our study provides a genetic basis for further elucidation of the characteristics and functional mechanisms of P. heimbachae as a conditionally pathogenic bacterium, as well as a direction for research into the mechanism of action of P. heimbachae infecting humans, extending knowledge of P. heimbachae as an important zoonotic pathogen.

Bacterium-like particles derived from probiotics: progress, challenges and prospects.

Bacterium-like particles (BLPs) are hollow peptidoglycan particles obtained from food-grade Lactococcus lactis inactivated by hot acid. With the advantage of easy preparation, high safety, great stability, high loading capacity, and high mucosal delivery efficiency, BLPs can load and display proteins on the surface with the help of protein anchor (PA), making BLPs a proper delivery system. Owning to these features, BLPs are widely used in the development of adjuvants, vaccine carriers, virus/antigens purification, and enzyme immobilization. This review has attempted to gather a full understanding of the technical composition, characteristics, applications. The mechanism by which BLPs induces superior adaptive immune responses is also discussed. Besides, this review tracked the latest developments in the field of BLPs, including Lactobacillus-derived BLPs and novel anchors. Finally, the main limitations and proposed breakthrough points to further enhance the immunogenicity of BLPs vaccines were discussed, providing directions for future research. We hope that further developments in the field of antigen delivery of subunit vaccines or others will benefit from BLPs.

The prevalence, genetic diversity and evolutionary analysis of cachavirus firstly detected in northeastern China.

Canine cachavirus is a novel parvovirus belonging to the genus Chaphamaparvovirus that was first detected in dogs in the United States. However, our knowledge of the prevalence and genetic characteristics of cachavirus is relatively limited. In this study, 325 canine fecal specimens collected from healthy and diarrheic dogs in northeastern China were screened with PCR. Twenty-two of the 325 (6.8%) samples were positive for cachavirus. The diarrhea samples showed high viral coinfection rates, and we detected coinfections with canine astrovirus (CaAstV) and cachavirus for the first time. A sequence analysis revealed that the Chinese cachavirus strains have point mutations in four consecutive amino acid codons relative to the original American strain. A codon usage analysis of the VP1 gene showed that most preferred codons in cachavirus were A- or T-ending codons, as in traditional canine parvovirus 2. A co-evolutionary analysis showed that cachavirus has undergone cospeciation with its hosts and has been transmitted among different host species. Our findings extend the limited cachavirus sequences available, and provide detailed molecular characterization of the strains in northeastern China. Further epidemiological surveillance is required to determine the significance and evolution of cachavirus.

Rosemary extract improves egg quality by altering gut barrier function, intestinal microbiota and oviductal gene expressions in late-phase laying hens.

BACKGROUND: Rosemary extract (RE) has been reported to exert antioxidant property. However, the application of RE in late-phase laying hens on egg quality, intestinal barrier and microbiota, and oviductal function has not been systematically studied. This study was investigated to detect the potential effects of RE on performance, egg quality, serum parameters, intestinal heath, cecal microbiota and metabolism, and oviductal gene expressions in late-phase laying hens. A total of 210 65-week-old "Jing Tint 6" laying hens were randomly allocated into five treatments with six replicates and seven birds per replicate and fed basal diet (CON) or basal diet supplemented with chlortetracycline at 50 mg/kg (CTC) or RE at 50 mg/kg (RE50), 100 mg/kg (RE100), and 200 mg/kg (RE200). RESULTS: Our results showed that RE200 improved (P < 0.05) Haugh unit and n-6/n-3 of egg yolk, serum superoxide dismutase (SOD) compared with CON. No significant differences were observed for Haugh unit and n-6/n-3 of egg yolk among CTC, RE50, RE100 and RE200 groups. Compared with CTC and RE50 groups, RE200 increased serum SOD activity on d 28 and 56. Compared with CON, RE supplementation decreased (P < 0.05) total cholesterol (TC) level. CTC, RE100 and RE200 decreased (P < 0.05) serum interleukin-6 (IL-6) content compared with CON. CTC and RE200 increased jejunal mRNA expression of ZO-1 and Occludin compared with CON. The biomarkers of cecal microbiota and metabolite induced by RE 200, including Firmicutes, Eisenbergiella, Paraprevotella, Papillibacter, and butyrate, were closely associated with Haugh unit, n-6/n-3, SOD, IL-6, and TC. PICRUSt2 analysis indicated that RE altered carbohydrate and amino acid metabolism of cecal microbiota and increased butyrate synthesizing enzymes, including 3-oxoacid CoA-transferase and butyrate-acetoacetate CoA-transferase. Moreover, transcriptomic analysis revealed that RE200 improved gene expressions and functional pathways related to immunity and albumen formation in the oviductal magnum. CONCLUSIONS: Dietary supplementation with 200 mg/kg RE could increase egg quality of late-phase laying hens via modulating intestinal barrier, cecal microbiota and metabolism, and oviductal function. Overall, RE could be used as a promising feed additive to improve egg quality of laying hens at late stage of production.

Genomic characterization and phylogenetic analysis of Aleutian mink disease virus identified in a sudden death mink case.

Aleutian mink disease (AMD) is one of the most serious diseases in minks worldwide, it brings tremendous financial losses in mink farming. AMD virus (AMDV) has unusually high genetic diversity, its genomic structure remains unclear. In 2014, sudden death of breeding minks was occurred in northeast China. After clinical signs evaluation and virus isolation, AMDV was identified in all sudden death minks, we investigated the complete genomic sequence of AMDV-LM isolated from the sudden death case. The full-genome sequence of AMDV-LM was 7 nucleotides (nts) or 8 nts longer than isolates AMDV-BJ and AMDV-G. AMDV-LM contained two unique nucleotide changes in VP2 (G79T, T710C), which led to two amino acid changes G27W and L237S. For NS1, some unique point mutations, such as A374C, A428C, A463C, and T476A were found and resulted in four unique amino acid mutations at N24V, H125P, V143P, K155Q, and V159N, respectively. The predicted secondary structure of the 5' terminal of AMDV-LM formed a large bubble formation near the 5' end, which affected the stability of the U-shaped hairpin. Phylogenetic analysis demonstrated that AMDV-LM was closely related to Chinese isolates and confirmed that AMDV strains circulating in China had different origins of ancestors. This study was first to investigate the association of sudden death of adult breeding minks with AMDV infection. Our findings provide useful suggestions for evaluation of the pathogenic potential of AMDV, additional details on AMDV genome characterization were also presented. Future work should focus on the importance of AMDV-LM strain in mink infection.

Multiple genotypes infection and molecular characterization of Torque teno neovison virus: A novel Anelloviridae of mink in China.

A novel Torque teno neovison virus (TTVs) was identified in specimens collected from dead mink during an outbreak of the Aleutian mink disease virus. Eighteen complete genomic sequences were obtained, ranging from 2109 to 2158 nucleotides in length and consisting of an untranslated region and three open reading frames. The genomic organization of mink TTVs is similar to previously reported anelloviruses. However, the deduced amino acid sequence of its ORF1 protein shows genetic diversity compared to related anelloviruses, suggesting that it represents a putative new species within the Anelloviridae family. This study provides a detailed molecular characterization of the novel mink anelloviruses, including its codon usage pattern, origin, and evolution. Analysis of the viral genomic sequences reveals the existence of multiple genotypes of co-infection. Principal component analysis and phylogenetic trees confirm the coexistence of multiple genotypes. Furthermore, the codon usage analyses indicate that mink TTVs have a genotype-specific codon usage pattern and show a low codon usage bias. Host-specific adaptation analysis suggests that TTVs are less adapted to mink. The possible origin and evolutionary history of mink TTVs were elucidated. Mink TTVs was genetically closely related to giant panda anellovirus, representing a new species. The observed incongruence between the phylogenetic history of TTVs and that of their hosts suggests that the evolution of anellovirus is largely determined by cross-species transmission. The study provides insights into the co-infection and genetic evolution of anellovirus in China.