Swine influenza virus triggers ferroptosis in A549 cells to enhance virus replication.

BACKGROUND: Recently, Influenza A virus (IAV) has been shown to activate several programmed cell death pathways that play essential roles in host defense. Indeed, cell death caused by viral infection may be mediated by a mixed pattern of cell death instead of a certain single mode. Ferroptosis is a novel form of regulated cell death (RCD) that is mainly mediated by iron-dependent lipid peroxidation. Based on the proteomic data, we wondered whether IAV causes ferroptosis in host cells. METHOD: In this study, a quantitative proteomics approach based on an iTRAQ combined with LC-MS/MS was used to profile proteins expressed in A549 cells infected with H1N1 swine influenza virus (SIV). Meanwhile, we measured the intracellular iron content, reactive oxygen species (ROS) release and lipid peroxidation in response to SIV infection. Finally, a drug experiment was conducted to investigate the effects of ferroptosis on modulating SIV survival. RESULTS: The bioinformatics analysis revealed several proteins closely relevant to iron homeostasis and transport, and the ferroptosis signaling pathway are highly enriched in response to SIV infection. In our experiment, aberrant expression of iron-binding proteins disrupted labile iron uptake and storage after SIV infection. Meanwhile, SIV infection inhibited system the Xc-/GPX4 axis resulting in GSH depletion and the accumulation of lipid peroxidation products. Notably, cell death caused by SIV as a result of iron-dependent lipid peroxidation can be partially rescued by ferroptosis inhibitor. Additionally, blockade of the ferroptotic pathway by ferrostatin-1 (Fer-1) treatment decreased viral titers and inflammatory response. CONCLUSIONS: This study revealed a new mode of cell death induced by IAV infection, and our findings might improve the understanding of the underlying mechanism involved in the interaction of virus and host cells.

Coinfection with PEDV and BVDV induces inflammatory bowel disease pathway highly enriched in PK-15 cells.

BACKGROUND: From the 1078 diarrhea stools tested in our survey from 2017 to 2020 in local area of China, PEDV was the key pathogen that was closely related to the death of piglets with diarrhea. In addition, coinfection of PEDV-positive samples with BVDV reached 17.24%. Although BVDV infection in swine is typically subclinical, the effect of PEDV and BVDV coinfection on disease severity and the potential molecular mechanism of coinfection with these two viruses remain unknown. METHODS: In this study, we developed a model of coinfection with porcine epidemic diarrhea virus (PEDV) and bovine viral diarrhea virus (BVDV) in PK15 cells, and a tandem mass tag (TMT) combined with LC-MS/MS proteomic approach was used to identify differential protein expression profiles. Additionally, we performed drug experiments to explore the inflammatory response induced by PEDV or BVDV mono- or coinfection. RESULTS: A total of 1094, 1538, and 1482 differentially expressed proteins (DEPs) were identified upon PEDV monoinfection, BVDV monoinfection and PEDV/BVDV coinfection, respectively. KEGG pathway analysis revealed that PEDV and BVDV coinfection led to a highly significantly enrichment of the inflammatory bowel disease (IBD) pathway. In addition, the NF-κB signaling pathway was more intensively activated by PEDV and BVDV coinfection, which induced higher production of inflammatory cytokines, than PEDV or BVDV monoinfection. CONCLUSIONS: Our study indicated that cattle pathogens might play synergistic roles in the pathogenesis of porcine diarrhea, which might also improve our understanding of the pathogenesis of multiple infections in diarrhea.

Long Noncoding RNAs Testis Development Related Gene 1 Aggravates Transforming Growth Factor-ß1-Induced Fibrogenesis and Inflammatory Response of Cardiac Fibroblasts Via miR-605-3p/Tumor Necrosis Factor Receptor Superfamily-21 Axis.

ABSTRACT: Heart failure is mainly caused by a decline in the systolic function of the heart. Long noncoding RNAs are related to cardiac diseases. This study aimed to explore the effects of long noncoding RNAs testis development related gene 1 (TDRG1) on the fibrogenesis and inflammatory response of transforming growth factor-beta1 (TGF-ß1)-stimulated human cardiac fibroblasts (HCFs). Levels of proinflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Reverse-transcription quantitative polymerase chain reaction was applied to reveal the expression levels of TDRG1, miR-605-3p, and tumor necrosis factor receptor superfamily (TNFRSF21). Western blot analysis was prepared to detect protein levels of TNFRSF21 and fibrosis-related genes. Luciferase reporter assay was conducted for confirming the interaction between miR-605-3p and TDRG1/TNFRSF21. We found that TGF-ß1-stimulated HCFs showed high concentrations of proinflammatory cytokines and increased protein levels of fibrosis-related genes, suggesting the dysfunctions of TGF-ß1-stimulated HCFs. In addition, TDRG1 was upregulated in TGF-ß1-stimulated HCFs. We found that interfering with TDRG1 alleviated dysfunctions of TGF-ß1-stimulated HCFs. Moreover, TDRG1 bound with miR-605-3p. MiR-605-3p exerted the antifibrogenic and anti-inflammatory effects in TGF-ß1-treated HCFs. As a target gene of miR-605-3p, TNFRSF21 reversed the antifibrogenic and anti-inflammatory effects of TDRG1 knockdown in TGF-ß1-treated HCFs. Overall, our study confirmed that TDRG1 aggravates fibrogenesis and inflammatory response in TGF-ß1-treated HCFs via the miR-605-3p/TNFRSF21 axis.

Molecular characterization of a porcine sapelovirus strain isolated in China.

Porcine sapelovirus (PSV) infections have been associated with a wide spectrum of symptoms, ranging from asymptomatic infection to clinical signs including diarrhoea, pneumonia, reproductive disorders, and polioencephalomyelitis. Although it has a global distribution, there have been relatively few studies on PSV in domestic animals. We isolated a PSV strain, SHCM2019, from faecal specimens from swine, using PK-15 cells. To investigate its molecular characteristics and pathogenicity, the genomic sequence of strain SHCM2019 was analysed, and clinical manifestations and pathological changes occurring after inoculation of neonatal piglets were observed. The virus isolated using PK-15 cells was identified as PSV using RT-PCR, transmission electron microscopy (TEM), and immunofluorescence assay (IFA). Sequencing results showed that the full-length genome of the SHCM2019 strain was 7,567 nucleotides (nt) in length, including a 27-nucleotide poly(A) tail. Phylogenetic analysis demonstrated that this virus was a PSV isolate belonging to the Chinese strain cluster. Recombination analysis indicated that there might be a recombination breakpoint upstream of the 3D region of the genome. Pathogenicity experiments demonstrated that the virus isolate could cause diarrhoea and pneumonia in piglets. In breif, a recombinant PSV strain, SHCM2019, was isolated and shown to be pathogenic. Our results may provide a reference for future research on the pathogenic mechanism and evolutionary characteristics of PSV.

Development and neutralization analysis of recombinant BVDVs expressing a CSF single chain antibody in vitro.

Although the immunization against swine fever (SF) is compulsory in China, it has still emerged in several areas at times. Herein, this study was conducted to develop an antibody vaccine which can clear the classical swine fever virus (CSFV) immediately after the pathogen invasion. Bovine viral diarrhoea virus (BVDV) infectious cDNA clone pASH28 was used to express a single-chain fragment variable (scFv) antibody against CSFV (CSFV/scFv) by reverse genetic technique. CSFV/scFv was inserted at the N-terminus of the C or Erns gene, generating two rBVDVs (rBVDV/C-CSFV/scFv and rBVDV/Erns-CSFV/scFv). Although both the rBVDVs could stably propagate on MDBK cells, different cellular characteristics existed. Obvious green fluorescence against the CSFV/scFv antibody could be visual on the cytomembrane or outside of the cells infected with rBVDV/Erns-CSFV/scFv, while much weaker fluorescence was observed in rBVDV/C-CSFV/scFv - infected cells. The CSFV/scFv antibodies induced by the two rBVDVs could recognize CSFV, but the rBVDV/Erns-CSFV/scFv induced stronger viral neutralization reaction. It was speculated that the neutralization activity might be associated with the expression location of CSFV/scFv antibody. The datas in this study provide evidence that rBVDV/Erns-CSFV/scFv may be engineered as a new antibody vaccine candidate against CSFV in the future.

The tyrosine 73 and serine 83 dephosphorylation of H1N1 swine influenza virus NS1 protein attenuates virus replication and induces high levels of beta interferon.

BACKGROUND: Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated. METHOD: In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway. RESULTS: We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-ß expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-ß and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-ß response upon rSIV NS1 Y73F and rSIV NS1 S83A infection. CONCLUSION: We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.

Effects of the S42 residue of the H1N1 swine influenza virus NS1 protein on interferon responses and virus replication.

BACKGROUND: The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. In the avian influenza virus or human influenza virus, specific amino acids of NS1 have been shown to be important for the virus to antagonize host antiviral defenses and promote viral replication. However, little research has been reported regarding the swine influenza virus (SIV) NS1 protein. METHODS: To study the effects of the key amino acids of NS1, we rescued NS1 mutants (S42P, D92E, and S42P/D92E) of the A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability and cytokine production as well as the intracellular localization in cultured cells. RESULTS: We found that the S42P and D92E mutation displayed no changes on NS1 nuclear localization. The S42P (but not D92E) mutation suppressed protein synthesis and reduced virus growth properties, and there was an inability to antagonize host cell interferon production and IRF3 activation, which led to high levels of IFN-α and IFN-ß production. CONCLUSION: We conclude that the S42 residue of the NS1 of the A/swine/Shanghai/3/2014(H1N1) strain is the key amino acid in regulating the host IFN response by blocking the activation of IRF3 and thus facilitates virus replication.

Exogenous administration of mitochondrial DNA promotes ischemia reperfusion injury via TLR9-p38 MAPK pathway.

Previous studies have shown a role of mitochondrial DNA (mtDNA) in innate immunity. However, the specific role of mtDNA in acute myocardial infarction remains elusive. This study was designed to examine the damaging effect of mtDNA on cardiomyocytes. H9c2s cells were incubated with purified mtDNA or nuclear DNA with or without pretreatment by chloroquine, an inhibitor of Toll-like receptor 9(TLR9). The cell viability was tested by MTT. To demonstrate the toxicity of mtDNA, mtDNA fragments were injected into rats 10 min before ischemia for 30 min and reperfusion for 24 h. Infarct size was measured by TTC staining. Apoptosis of myocardium was detected by TUNEL staining and caspase-3 activity. The levels of TLR9, p-p38 MAPK, and p38 MAPK were detected by western blotting. The results showed that exogenous mtDNA reduced the viability of H9c2s cells and induced TLR9 expression, caspase 3 activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation. However, these effects were inhibited by chloroquine. In contrast, nuclear DNA did not have these effects. Intravenous injection of mtDNA into rats aggravated ischemia-reperfusion injury and increased infarction area through TLR9-p38 MAPK activation. We concluded that mtDNA released into the circulation by AMI may has detrimental effect on myocardium through aggravating ischemia-reperfusion injury via TLR9-p38 MAPK pathway.

Antiestrogenic Activity of Triptolide in Human Breast Cancer Cells MCF-7 and Immature Female Mouse.

Preclinical Research To investigate the antiestrogenic activity of triptolide in human breast cancer cell line MCF-7 and immature female C57BL/6 mouse. The effects of triptolide on cell proliferation, cell cycle, and the expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) were examined in MCF-7 cells. In vivo antiestrogenic effects of triptolide were observed after cotreatment of mice with E2 and triptolide for 4 days. Triptolide dose- and time-dependently inhibited cell growth in untreated or E2 -treated MCF-7 cells, which was associated with increased S phase arrest. Furthermore, triptolide down regulated the expression of ERα and PR in cells. The expression of ERα and PR in combined group of triptolide with E2 was much higher than that of triptolide alone. Triptolide decreased the E2 -induced uterine weight in mice, while triptolide alone had no effect. Triptolide treatment (90 µg/kg) resulted in extensive degeneration and necrosis of uterine epithelial cells, whereas the same concentration of triptolide in combination with E2 caused morphologic changes in epithelial cells from simple columnar to ellipse, without destruction. Triptolide showed antiestrogenic activity in vitro and in vivo, and the down regulation of ERα and PR expression may be its underlying mechanisms. Drug Dev Res 78 : 164-169, 2017. © 2017 Wiley Periodicals, Inc.

The Distribution of 18 Enterotoxin and Enterotoxin-Like Genes in Staphylococcus aureus Strains from Different Sources in East China.

The distribution of 18 staphylococcal enterotoxin (SE) or SE-like (SEl) genes in Staphylococcus aureus strains from different sources in east China was investigated. Among all 496 S. aureus strains, 291 strains carried one or more SE genes. The more frequently occurred genes were sea, seb, seg, selk, sell, selm, selo, and seq; the less frequent occurred genes were sec, selj, and ser. The classic SE genes and the enterotoxin gene cluster (egc) (seg, sei, selm, seln, selo, and/or selu) accounted for 25.67% and 61.68% of all detected genes, respectively. There were three gene clusters (egc, sea-sek-seq, and sed-sej-ser), of which the egc cluster was the important one that could generate novel complexes, and the sea-sek-seq cluster was a close relative to the hospital-acquired methicillin-resistant S. aureus. The SE gene distributions were different among strains of different sources and formed diverse toxin gene profiles. The human- and foodborne-origin strains harbored classic and novel SE and SEl genes, whereas animal-origin strains harbored egc and other novel SE and SEl genes mainly. The foodborne- and human-origin strains were the main dangerous factors of classic staphylococcal foodborne poisoning, whereas the strains (especially from animals) that carried egc and other novel genes mainly should be new potential dangerous factors for food safety.

Nuclear localization of the p17 protein of avian reovirus is correlated with autophagy induction and an increase in viral replication.

p17 is a nonstructural protein of avian reovirus (ARV) that induces autophagy in infected cells. In the present study, we investigated the effect of p17 and its nuclear localization signal (NLS) on autophagy and viral replication. When Vero cells and DF1 cells were transfected with mutant p17 in which lysine (K) at position 122 and arginine (R) at position 123 were mutated to alanine (A), the expression level of LC3 II decreased dramatically after transfection. The expression of the polypeptide encompassing the first 103 amino acids of p17, a region that did not contain the NLS, did not have a significant effect on autophagy. Moreover, when cells overexpressing mutant p17 were infected with the ARV GX2010/1 strain, the viral titer was significantly decreased compared with the expression of wild-type p17. In general, the NLS of p17 facilitates the induction of autophagy and is correlated with an increase in virus production.

Autophagy inhibitors reduce avian-reovirus-mediated apoptosis in cultured cells and in chicken embryos.

Avian reovirus (ARV)-induced apoptosis contributes to the pathogenesis of reovirus in infected chickens. However, methods for effectively reducing ARV-triggered apoptosis remain to be explored. Here, we show that pretreatment with chloroquine (CQ) or E64d plus pepstatin A decreases ARV-mediated apoptosis in chicken DF-1 cells. By acting as autophagy inhibitors, CQ and E64d plus pepstatin A increase microtubule-associated protein 1 light chain 3-II (LC3II) accumulation in ARV-infected cells, which results in decreased ARV protein synthesis and virus yield and thereby contributes to the reduction of apoptosis. Furthermore, ARV-mediated apoptosis in the bursa, heart and intestines of chicken embryos is attenuated by CQ and E64d plus pepstatin A treatment. Importantly, treatment with these autophagy inhibitors increases the survival of infected chicken embryos. Together, our data suggest that pharmacological inhibition of autophagy might represent a novel strategy for reducing ARV-mediated apoptosis.

Circulating Cell Free Mitochondrial DNA is a Biomarker in the Development of Coronary Heart Disease in the Patients with Type 2 Diabetes.

BACKGROUND: Circulating cell-free mtDNA (ccf-mtDNA) is presumably derived from injured tissue or cells in the body and has been suggested to be a potential biomarker in several diseases. In the present study, we investigated whether ccf-mtDNA levels could serve as a biomarker of diabetic patients without Coronary Heart Disease (CHD) and correlate with their incidence to develop CHD. METHODS: Ccf-mtDNA levels in plasma and the traditional CHD risk factors were determined in 50 type 2 diabetes (DM) with CHD and 50 DM without CHD patients and 50 age- and sex-matched healthy controls. MtDNA levels were assessed by measuring the NADH dehydrogenase 1 gene using quantitative real-time PCR. ROC curve analyses of plasma mtDNA in DM with or without CHD were also determined. Multivariate logistic regression analyses were conducted to determine the correlation between the mtDNA levels and traditional CHD risk factors. RESULTS: The plasma ccf-mtDNA levels and CRP were significantly elevated in DM compared with healthy controls, and a consistent increase of mtDNA in diabetes with CHD compared to those without CHD. The area under the ROC curves of mtDNA in DM patients versus DM patients with CHD and healthy controls was 0.843 and 0.898, respectively. Correlation analyses showed that the mtDNA levels were significantly correlated with that of C-reactive protein (CRP) in patients with CHD. CONCLUSIONS: Ccf-mtDNA was elevated in type 2 diabetes with CHD and correlated with CRP levels. Both may be considered a prognostic biomarker of CHD development.

Toll-like receptor 3 inhibits Newcastle disease virus replication through activation of pro-inflammatory cytokines and the type-1 interferon pathway.

Newcastle disease virus (NDV) is an avian paramyxovirus that can selectively replicate in and destroy human tumor cells. In this report, we demonstrate that NDV infection in HeLa cells leads to the activation of the pattern recognition Toll-like receptor 3 (TLR3). Overexpression of TLR3 enhanced the activity of the IFN-ß promoter and the transcription factor NF-kappa B (NF-κB), thereby decreasing viral protein synthesis and the virus titer. In addition, the reduction of endogenous TLR3 by small interfering RNA (siRNA) increased NDV replication. Similar anti-NDV effects were observed in DF-1 chicken fibroblast cells with the exogenous expression of chicken TLR3 (cTLR3). Immunofluorescence staining of HeLa cells indicated that the dsRNA generated during NDV replication colocalized with TLR3 in punctate subcellular structures. Altogether, our results strongly suggest that TLR3 actively participates in the recognition of the innate pro-inflammatory response after NDV infection and leads to the consequent antiviral cytokine/interferon secretion.

NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light-Chain Kinase (ERK/MLCK) Pathway.

Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

Microbial Community and Metabolome Analysis of the Porcine Intestinal Damage Model Induced by the IPEC-J2 Cell Culture-Adapted Porcine Deltacoronavirus (PDCoV) Infection.

This study was conducted to elucidate the intestinal damage induced by the IPEC-J2 cell culture-passaged PDCoV. The results showed that PDCoV disrupted the intestinal structure and increased intestinal permeability, causing abnormalities in mucosal pathology. Additionally, PDCoV induced an imbalance in the intestinal flora and disturbed its stability. Microbial community profiling revealed bacterial enrichment (e.g., Proteobacteria) and reduction (e.g., Firmicutes and Bacteroidetes) in the PDCoV-inoculated piglet model. In addition, metabolomics analysis indicated that 82 named differential metabolites were successfully quantified, including 37 up-regulated and 45 down-regulated metabolites. Chenodeoxycholic acid, sphingosine, and oleanolic aldehyde levels were reduced in PDCoV-inoculated piglets, while phenylacetylglycine and geranylgeranyl-PP levels were elevated. Correlation analysis indicated a negative correlation between Escherichia-Shigella and choline, succinic acid, creatine, phenyllactate, and hippuric acid. Meanwhile, Escherichia-Shigella was positively correlated with acetylcholine, L-Glutamicacid, and N-Acetylmuramate. Roseburia, Lachnospiraceae_UCG-010, Blautia, and Limosilactobacillus were negatively and positively correlated with sphingosine, respectively. These data suggested PDCoV-inoculated piglets exhibited significant taxonomic perturbations in the gut microbiome, which may result in a significantly altered metabolomic profile.

Pollution levels and ecological risks of PPCPs in water and sediment samples of Danjiangkou Reservoir.

The concentrations and distribution patterns of three typical pharmaceuticals and personal care products (PPCPs) in water and sediment samples obtained from Danjiangkou Reservoir during two seasonal sampling periods were studied to determine their impact on water quality. The temporal and spatial variations in concentrations measured were analyzed and related to ecological risks with data obtained during the mean-flow period (in June) and the dry period (in November). We found a high detection rate of ketoprofen (KTP) in water samples from Danjiangkou Reservoir; the concentrations ranged from not detected (ND) to 46.80 ng/L with the highest values measured in the Hanku tributary samples followed by the samples collected in the main body of Danjiangkou Reservoir. The KTP concentrations in the Danku tributary samples were the lowest measured in this study. In addition, the concentrations of KTP in the Shending River, Sihe River, Jianghe River, Guanshan River, and Jianhe River water samples were relatively high in the mean-flow period. The water sample detection rates and concentrations of triclosan (TCS) and triclocarban (TCC) were low in both the mean-flow period and the dry period. All three kinds of PPCPs were detected in the sediment samples with the concentrations of KTP, TCS, and TCC ranging from 0.76 to 7.89 µg/kg, 0.01 to 0.59 µg/kg, and 0.01 to 11.36 µg/kg, respectively. Overall, the concentrations of the three measured PPCPs in the water and sediment samples were all relatively low compared to results reported in the recent literature. The dry period concentrations of PPCPs in the water samples were lower than the concentrations measured in the mean-flow period. However, dry period concentrations were higher in the sediment samples compared to those in the mean-flow period samples. Our interpretation of the spatial and temporal patterns of PPCPs in Danjiangkou Reservoir suggests that these compounds were likely mainly derived from wastewater discharge in the upper reaches of the reservoir. The risk quotient (RQ) method was used for an ecological risk assessment of the detected PPCPs in this study. We found that TCS in water and sediment posed medium ecological risks to algae at different times of the year. In view of the extreme importance of water safety in Danjiangkou Reservoir, the ecological risks of PPCPs require additional attention.

Genomic Evolution and Selective Pressure Analysis of a Novel Porcine Sapovirus in Shanghai, China.

Porcine sapovirus (PoSaV) is one of the most significant pathogens causing piglet diarrhea, and one with limited genetic characterization. In this study, the prevalence, infection pattern, and genetic evolution of porcine sapovirus were elucidated in detail. The positive rate of PoSaV was 10.1% (20/198), with dual, triple, and quadruple infections of 45%, 40%, and 5%, respectively. To further explore the viral composition in the PoSaV-positive diarrhea feces, metagenomic sequencing was carried out. The results confirmed that RNA viruses accounted for a higher proportion (55.47%), including the two primary viruses of PoSaV (21.78%) and porcine astrovirus (PAstV) (24.54%) in the tested diarrhea feces samples. Afterward, a full-length sequence of the PoSaV isolate was amplified and named SHCM/Mega2023, and also given the identifier of GenBank No. PP388958. Phylogenetic analysis identified the prevalent PoSaV strain SHCM/Mega2023 in the GIII genogroup, involving a recombinant event with MK962338 and KT922089, with the breakpoint at 2969-5132 nucleotides (nt). The time tree revealed that the GIII genogroup exhibits the widest divergence time span, indicating a high likelihood of viral recombination. Moreover, SHCM/Mega2023 had three nucleotide "RPL" insertions at the 151-153 nt site in the VP2 gene, compared to the other GIII strains. Further selective pressure calculations demonstrate that the whole genome of the SHCM/Mega2023 strain was under purifying selection (dN/dS < 1), with seven positively selected sites in the VP1 protein, which might be related to antigenicity. In conclusion, this study presents a novel genomic evolution of PoSaV, offering valuable insights into antigenicity and for vaccine research.

A ferritin nanoparticle vaccine based on the hemagglutinin extracellular domain of swine influenza A (H1N1) virus elicits protective immune responses in mice and pigs.

Introduction: Swine influenza viruses (SIVs) pose significant economic losses to the pig industry and are a burden on global public health systems. The increasing complexity of the distribution and evolution of different serotypes of influenza strains in swine herds escalates the potential for the emergence of novel pandemic viruses, so it is essential to develop new vaccines based on swine influenza. Methods: Here, we constructed a self-assembling ferritin nanoparticle vaccine based on the hemagglutinin (HA) extracellular domain of swine influenza A (H1N1) virus using insect baculovirus expression vector system (IBEVS), and after two immunizations, the immunogenicities and protective efficacies of the HA-Ferritin nanoparticle vaccine against the swine influenza virus H1N1 strain in mice and piglets were evaluated. Results: Our results demonstrated that HA-Ferritin nanoparticle vaccine induced more efficient immunity than traditional swine influenza vaccines. Vaccination with the HA-Ferritin nanoparticle vaccine elicited robust hemagglutinin inhibition titers and antigen-specific IgG antibodies and increased cytokine levels in serum. MF59 adjuvant can significantly promote the humoral immunity of HA-Ferritin nanoparticle vaccine. Furthermore, challenge tests showed that HA-Ferritin nanoparticle vaccine conferred full protection against lethal challenge with H1N1 virus and significantly decreased the severity of virus-associated lung lesions after challenge in both BALB/c mice and piglets. Conclusion: Taken together, these results indicate that the hemagglutinin extracellular-based ferritin nanoparticle vaccine may be a promising vaccine candidate against SIVs infection.

The lncRNA HCG4 regulates the RIG-I-mediated IFN production to suppress H1N1 swine influenza virus replication.

Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor that allows the virus to replicate efficiently by suppressing host innate immune responses. Previously, we demonstrated that the serine (S) at position 42 of NS1 in H1N1 swine influenza virus (SIV) is a critical residue in interferon (IFN) resistance, thus facilitating viral infections. Here, by lncRNA-seq, a total of 153 differentially expressed lncRNAs were identified, and the lncRNA HCG4 was selected due to its significantly higher expression after infection with the NS1 S42P mutant virus. Overexpression of HCG4 enhanced IFN-ß production and suppressed SIV infection, highlighting the potential antiviral activity of HCG4 against SIV. Further investigation suggested that HCG4 served as a positive feedback mediator for RIG-I signaling. It alleviated the inhibitory effect on RIG-I K63-linked ubiquitination by NS1 protein, thereby resulting in an increase in RIG-I-mediated IFN production. Taken together, our findings demonstrate that HCG4 modulates the innate immune response to SIV infection through K63-linked RIG-I ubiquitination, providing insights into the role of lncRNAs in controlling viral infections.