Enhanced in vivo antiviral activity against pseudorabies virus through transforming gallic acid into graphene quantum dots with stimulation of interferon-related immune responses.

With the urgent need for antiviral agents, antiviral materials with high biocompatibility and antiviral effects have attracted a lot of attention. In this study, gallic acid, a natural polyphenolic compound, was transformed into biocompatible graphene quantum dots (GAGQDs) which exhibit enhanced antiviral activity against pseudorabies virus (PRV). The as-prepared GAGQDs inhibit PRV proliferation with a 104-fold reduction in viral titers. Investigation of the antiviral mechanism revealed that GAGQDs inhibit the adsorption, invasion and replication of PRV infection. Treatment with GAGQDs regulates the expression levels of interferon-related antiviral proteins, including mitochondrial antiviral-signaling protein (MAVS), signal transducer and activator of transcription 1 (STAT1) and 2',5'-oligoadenylate synthetase 1 (OAS1), suggesting that GAGQDs can stimulate innate antiviral immune responses, resulting in enhanced antiviral effects. More importantly, GAGQD treatments alleviate clinical symptoms and reduce mortality in PRV-infected mice. Our results reveal the enhanced therapeutic effects of GAGQDs against PRV infection in vitro and in vivo, suggesting the potential of GAGQDs as a promising novel antiviral agent.

Integrative transcriptomic and metabolomic analysis in mice reveals the mechanism by which ginseng stem-leaf saponins enhance mucosal immunity induced by a porcine epidemic diarrhea virus vaccination.

Porcine epidemic diarrhea virus (PEDV) is a main cause of severe enteric disease in piglets, leading to millions of dollars lost annually in the global pig industry. Parenteral vaccination is limited in generating sufficient mucosal immunity, which is crucial for early defense against PEDV. Here, we orally administered ginseng stem-leaf saponins (GSLS) to mice before parenteral vaccination and found that GSLS significantly enhanced the phagocytosis of dendritic cells, promoted the activities of CD4+ T cells and increased PEDV-specific IgA antibodies in the intestinal mucosa. Transcriptomic results showed that the altered genes following GSLS treatment were mostly related to the immune response and metabolism. In addition, integrated analysis of the transcriptome and metabolome revealed that the mechanism by which GSLS enhances mucosal immunity may be associated with progesterone-related pathways. Further studies are needed to explore the detailed molecular mechanisms.

Early Oral Administration of Ginseng Stem-Leaf Saponins Enhances the Peyer's Patch-Dependent Maternal IgA Antibody Response to a PEDV Inactivated Vaccine in Mice, with Gut Microbiota Involvement.

Neonatal piglets during the first week of life are highly susceptible to porcine epidemic diarrhoea virus (PEDV) infection, with mortality rates reaching 80-100%. Passive lactogenic immunity remains the most effective way to protect neonates from infection. Although safe, inactivated vaccines provide little or no passive protection. Here, we administered ginseng stem-leaf saponins (GSLS) to mice before parenteral immunization with an inactivated PEDV vaccine to investigate the effect of GSLS on the gut-mammary gland (MG)-secretory IgA axis. Early oral GSLS administration potently increased PEDV-specific IgA plasma cell generation in the intestine, facilitated intestinal IgA plasma cell migration to the MG by enhancing the chemokine receptor (CCR)10-chemokine ligand (CCL)28 interaction, and ultimately promoted specific IgA secretion into milk, which was dependent on Peyer's patches (PPs). Additionally, GSLS improved the gut microbiota composition, especially increasing probiotic abundance, and these microflora members promoted the GSLS-enhanced gut-MG-secretory IgA axis response and were regulated by PPs. In summary, our findings highlight the potential of GSLS as an oral adjuvant for PEDV-inactivated vaccines and provide an attractive vaccination strategy for lactogenic immunity induction in sows. Further studies are required to evaluate the mucosal immune enhancement efficacy of GSLS in pigs.

Dumbbell-like upconversion nanoparticles synthesized by controlled epitaxial growth for light-heat-color tri-modal sensing of carcinoembryonic antigen.

Accurate quantitative analysis of tumor markers in a wide linear range has important practical significance towards complex clinical samples in cancer identification and monitoring of tumor development stages, but remains challenging. Herein, three-layer dumbbell-like upconversion nanoparticles NaErF4:Tm@NaYF4@NaNdF4 (labeled as UCNPs) combined with G-quadruplex (G4) DNAzyme are reported for tri-modal sensing of carcinoembryonic antigen (CEA) in a wide range using upconversion luminescence (UCL), photothermal and catalysis signal readouts. Initially, dumbbell-like UCNPs were controlled synthesized by a three-dimensional epitaxial growth strategy through tuning the concentration of Nd precursors. After surface functionalization, G4zyme-UCNPs-cDNA/Apt-MB was subsequently fabricated by biotin-streptavidin interaction and DNA hybridization. Quantitative detection of CEA was achieved by competitive interaction and magnetic separation, and the intensities of tri-modal signals (light, heat and catalysis-based chrominance) of dissociative probes are linearly related to the concentration of CEA. The results showed that the tri-modal sensing method exhibited a wide linear range (0.005-2000 ng/mL) and low limit of detection (LOD) across three models: the luminescence model (0.005-50 ng/mL, LOD = 0.910 pg/mL), the catalysis model (10-1000 ng/mL, LOD = 0.387 ng/mL), and the temperature model (50-2000 ng/mL, LOD = 1.114 ng/mL). These findings suggest that the tri-modal sensing platform is suitable for use in the analysis of a wide range of complex and diverse clinical samples.

Immune Enhancement of Nanoparticle-Encapsulated Ginseng Stem-Leaf Saponins on Porcine Epidemic Diarrhea Virus Vaccine in Mice.

Porcine epidemic diarrhea virus (PEDV) causes severe enteric disease in pigs, particularly neonatal piglets. Current vaccines do not provide complete protection against PEDV. Ginseng stem-leaf saponins (GSLS), a promising oral adjuvant candidate, can improve intestinal immune responses in poultry and mice. However, its low stability limits further use. Poly lactic-co-glycolic acid (PLGA), a biocompatible and biodegradable nanoparticle, has been widely used in biomedicine for stable and targeted drug delivery. In this study, we developed GSLS-PLGA nanoparticles (GSLS-NPs) and evaluated the mucosal adjuvant efficacy in vitro and in vivo. GSLS-NPs significantly enhanced antigen internalization and pro-inflammatory cytokine secretion by DC2.4 cells. Mice orally administered GSLS-NPs before intramuscular inoculation generated CD11b+CD8α- and CD11b-CD103+ dendritic cells in the spleen and draining mesenteric lymph nodes, respectively, which are the types mainly responsible for antigen presentation. Additionally, enhanced neutralizing and non-neutralizing antibody responses and expanded activities of specific effector and memory CD4+ and CD8+ T cells were also observed in mice immunized with PEDV vaccines plus GSLS-NPs compared to mice receiving the vaccines alone. Furthermore, GSLS-NPs showed a good safety profile and presented great advantages over GSLS aqueous solution. Collectively, our results highlight the potential of GSLS-NPs as a mucosal adjuvant and provide an attractive vaccination strategy for combatting PEDV. Further study is required to evaluate the efficacy of this mucosal adjuvant in swine.

Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces, Eastern China.

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I-induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α-mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target.

High-resolution genome-wide association study and genomic prediction for disease resistance and cold tolerance in wheat.

KEY MESSAGE: High-resolution genome-wide association study (GWAS) facilitated QTL fine mapping and candidate gene identification, and the GWAS based genomic prediction models were highly predictive and valuable in wheat genomic breeding. Wheat is a major staple food crop and provides more than one-fifth of the daily calories and dietary proteins for humans. Genome-wide association study (GWAS) and genomic selection (GS) for wheat stress resistance and tolerance related traits are critical to understanding their genetic architecture for improvement of breeding selection efficiency. However, the insufficient marker density in previous studies limited the utility of GWAS and GS in wheat genomic breeding. Here, we conducted a high-resolution GWAS for wheat leaf rust (LR), yellow rust (YR), powdery mildew (PM), and cold tolerance (CT) by genotyping a panel of 768 wheat cultivars using genotyping-by-sequencing. Among 153 quantitative trait loci (QTLs) identified, 81 QTLs were delimited to ≤ 1.0 Mb intervals with three validated using bi-parental populations. Furthermore, 837 stress resistance-related genes were identified in the QTL regions with 12 showing induced expression by YR and PM pathogens. Genomic prediction using 2608, 4064, 3907, and 2136 pre-selected SNPs based on GWAS and genotypic correlations between the SNPs showed high prediction accuracies of 0.76, 0.73, and 0.78 for resistance to LR, YR, and PM, respectively, and 0.83 for resistance to cold damage. Our study laid a solid foundation for large-scale QTL fine mapping, candidate gene validation and GS in wheat.

Escherichia coli Heat-Labile Enterotoxin B Subunit Combined with Ginsenoside Rg1 as an Intranasal Adjuvant Triggers Type I Interferon Signaling Pathway and Enhances Adaptive Immune Responses to an Inactivated PRRSV Vaccine in ICR Mice.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen that has threatened the global swine industry for almost 30 years. Because current vaccines do not provide complete protection, exploration of new preventive strategies is urgently needed. Here, we combined a heat-labile enterotoxin B subunit of Escherichia coli (LTB) and ginsenoside Rg1 to form an intranasal adjuvant and evaluated its enhancement of immune responses in mice when added to an inactivated-PRRSV vaccine. The combination adjuvant synergistically elicited higher neutralizing and non-neutralizing (immunoglobulin G and A) antibody responses in the circulatory system and respiratory tract, and enhanced T and B lymphocyte proliferation, CD4+ T-cell priming, and cytotoxic CD4+ T cell activities in mononuclear cells from spleen and lung tissues when compared to the PRRSV vaccine alone, and it resulted in balanced Th1/Th2/Th17 responses. More importantly, we observed that the combination adjuvant also up-regulated type I interferon signaling, which may contribute to improvement in adaptive immune responses. These results highlight the potential value of a combined adjuvant approach for improving the efficacy of vaccination against PRRSV. Further study is required to evaluate the efficacy of this combined adjuvant in swine.

High-Resolution Genome-wide Association Study Identifies Genomic Regions and Candidate Genes for Important Agronomic Traits in Wheat.

Wheat (Triticum aestivum) is a major staple food crop worldwide. Genetic dissection of important agronomic traits is essential for continuous improvement of wheat yield to meet the demand of the world's growing population. We conducted a large-scale genome-wide association study (GWAS) using a panel of 768 wheat cultivars that were genotyped with 327 609 single-nucleotide polymorphisms generated by genotyping-by-sequencing and detected 395 quantitative trait loci (QTLs) for 12 traits under 7 environments. Among them, 273 QTLs were delimited to ≤1.0-Mb intervals and 7 of them are either known genes (Rht-D, Vrn-B1, and Vrn-D1) that have been cloned or known QTLs (TaGA2ox8, APO1, TaSus1-7B, and Rht12) that were previously mapped. Eight putative candidate genes were identified for three QTLs that enhance spike seed setting and grain size using gene expression data and were validated in three bi-parental populations. Protein sequence analysis identified 33 putative wheat orthologs that have high identity with rice genes in QTLs affecting similar traits. Large r2 values for additive effects observed among the QTLs for most traits indicated that the phenotypes of these identified QTLs were highly predictable. Results from this study demonstrated that significantly increasing GWAS population size and marker density greatly improves detection and identification of candidate genes underlying a QTL, solidifying the foundation for large-scale QTL fine mapping, candidate gene validation, and developing functional markers for genomics-based breeding in wheat.

Swine Promyelocytic Leukemia Isoform II Inhibits Pseudorabies Virus Infection by Suppressing Viral Gene Transcription in Promyelocytic Leukemia Nuclear Bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against alphaherpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to alphaherpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoforms sPML-II and -IIa, not sPML-I and -IVa, expressed in a sPML knockout cells inhibit PRV infection. Both the unique 7b region of sPML-II and the sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residues 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of alphaherpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an antiviral function but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.

Th1-biased immunoadjuvant effect of the recombinant B subunit of an Escherichia coli heat-labile enterotoxin on an inactivated porcine reproductive and respiratory syndrome virus antigen via intranasal immunization in mice.

Porcine reproductive and respiratory syndrome (PRRS) is one of the major swine diseases responsible for a significant challenge in the global swine industry. The current PRRS inactivated vaccine only confers limited protection against PRRSV. Thus, using an appropriate adjuvant via a suitable administration route may help improve vaccine efficacy. In this study, the recombinant B subunit of the Escherichia coli heat-labile enterotoxin rLTB, was highly expressed in Pichia pastoris, through high-density fermentation. rLTB intranasal adjuvant properties were evaluated on an inactivated PRRS antigen in mice. Compared to the group immunized with solely PRRS antigen, a dose of 50 µg rLTB remarkably raised antigen-specific IgA antibodies at mucosal sites, and increased serum IgG antibodies, preferentially the IgG2a and IgG2b subclasses. Further, rLTB induced increases in Th1- (IFN-γ and IL-12) and Th17 (IL-6) cytokine profiles, but had little effect on Th2 cytokine profiles (IL-4 and IL-10). Moreover, there were no overt toxicities associated with intranasal rLTB administration. Our data provide evidence that the rLTB produced by P. pastoris fermentation portrays low toxicity, and its intranasal adjuvant effect involves immune system modulation to a Th1 profile.

Association between iscR-based phylogeny, serovars and potential virulence markers of Haemophilus parasuis.

Haemophilus parasuis is an economically important bacterial pathogen of swine. Extensive genetic and phenotypic heterogeneity among H. parasuis strains have been observed, which hinders the deciphering of the population structure and its association with clinical virulence. In this study, two highly divergent clades were defined according to iron-sulphur cluster regulator (iscR)-based phylogeny analysis of 148 isolates. Clear separation of serovars and potential virulence markers (PVMs) were observed between the two clades, which are indicative of independent evolution of the two lineages. Previously suggested virulence factors showed no correlation with clinical virulence, and were probably clade or serovar specific genes emerged during different stage of evolution. PVMs profiles varied widely among isolates in the same serovar. Higher strain diversity in respect of PVMs was found for isolates from multi-strain infected farms than those from single strain infected ones, which indicates that multi-strain infection in one farm may increase the frequency of gene transfer in H. parasuis. Systemic isolates were more frequently found in serovar 13 and serovar 12, while no correlation between clinical virulence and iscR-based phylogeny was observed. It shows that iscR is a reliable marker for studying population structure of H. parasuis, while other factors should be included to avoid the interference of gene exchange of iscR between isolates. The two lineages of H. parasuis may have undergone independent evolution, but show no difference in clinical virulence. Wide distribution of systemic isolates across the entire population poses new challenge for development of vaccine with better cross-protection. Our study provides new information for better deciphering the population structure of H. parasuis, which helps understanding the extreme diversity within this pathogenic bacterium.

Complete Genome Sequence of Variant Porcine Epidemic Diarrhea Virus Strain ZJ/ZX2018-C10, Isolated in Zhejiang, China, in 2018.

We report here the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain ZJ/ZX2018-C10, isolated from infected piglets in Zhejiang Province, China. The genome sequence was highly similar to AH2012, a highly virulent Chinese PEDV strain. It will help in understanding the molecular and evolutionary characteristics of PEDV in China.

Identification of a novel Hemoplasma species from pigs in Zhejiang province, China.

Hemoplasmas belong to Mycoplasmataceae (Mollicutes: Mycoplasmatales) and are able to infect a broad range of mammalian species. We investigated prevalence of hemotropic mycoplasma species in pig farms in the region of Zhejiang by a PCR scheme using universal primers targeting 16S rRNA and RNase P RNA gene (rnpB). Representative positive samples from different farms were selected for sequencing of 16S rRNA and the 219bp rnpB gene fragments for phylogenetic analysis. Sequencing analysis of PCR products from first samples identified a novel hemoplasma species present in several pig farms in the region with highest nucleotide identity of 92% to Candidatus Mycoplasma turicensis. A duplex PCR assay was then designed for differential detection of the novel hemoplasma from Mycoplasma parvum/M. suis in field samples. Of 324 blood samples from clinically healthy pigs, 26.5% was positive for this novel hemoplasma species and 50% positive for M. suis/M. parvum, indicating that the novel hemotropic mycoplasma species were of considerably high prevalence in Zhejiang province, China.

Efficient construction of Haemophilus parasuis mutants based on natural transformation.

Studies on virulence factors and pathogenecity of Haemophilus parasuis have long been hindered by a lack of a consistent system for genetic manipulation. In this study, competence was induced by transferring H. parasuis from rich medium to starvation medium media-IV (M-IV) and iscR gene deficient mutants of H. parasuis were generated efficiently. Transformation frequency varied from 4.1 × 10-5 to 1.1 × 10-8 when using circular plasmid, and increased to about 2- to 31-fold when transformed using linearized plasmid. Allele replacement occurred efficiently in 6 strains, which are transformable using both circular and linearized pTRU, but not in another 2 strains which could only be transformed using linearized plasmid. The iscR mutants were stable for at least 20 passages in vitro. Haemophilus parasuis strains vary extensively in natural transformation efficiency and the method established here allows for transformation of a larger spectrum of strains with an easily accessed plasmid. This provides important tools for genetic manipulation of H. parasuis.

Les études sur les facteurs de virulence et la pathogénicité d'Haemophilus parasuis ont longtemps été limitées à cause de l'absence d'un système constant de manipulations génétiques. Dans la présente étude, la compétence a été induite en transférant H. parasuis d'un milieu de culture riche au milieu pauvre M-IV et les H. parasuis mutants déficients pour le gène iscR étaient générés efficacement. La fréquence de transformation variait de 4,1 × 10−5 à 1,1 × 10−8 lors de l'utilisation d'un plasmide circulaire, et augmentait d'un facteur variant de 2 à 31 lorsque la transformation utilisait un plasmide linéarisé. Le remplacement d'allèle est survenu efficacement chez 6 souches, qui étaient transformables en utilisant le pTRU circulaire ou linéarisé, mais pas chez 2 autres souches qui ne pouvaient être transformées qu'en utilisant le plasmide linéarisé. Les mutants iscR étaient stables pendant au moins 20 passages in vitro. Les souches d'H. parasuis varient énormément dans leur efficacité de transformation naturelle et la méthode développée ici permet la transformation d'un plus large spectre de souches avec un plasmide facilement accessible. Ceci fournit d'importants outils pour la manipulation génétique d'H. parasuis.(Traduit par Docteur Serge Messier).

Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities.

In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV(DLA) into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV(DLA) showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.

[Reliability of detecting SARS-CoV antibody for diagnosis of SARS].

OBJECTIVE: To discuss the reliability of SARS-CoV antibody detection for SARS diagnosis. METHODS: Using SARS-CoV ELISA kit to detect relevant antibody in fresh serum of healthy, fever, probable, and suspect cases. RESULTS: The positive rate is 0%, 40%, and 95% respectively in healthy, probable, and suspect cases. CONCLUSIONS: It is reliable to detect SARS-CoV antibody in late suspect patients, but there will be high false-positive result in ordinary fever cases.