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.

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.

Development of an indirect ELISA method based on the VP1 protein for detection of IgG antibodies against porcine sapelovirus.

Porcine sapelovirus (PSV) is a newly emerging enterovirus that is widely prevalent in China. Since there is no clinical serological testing for PSV, the objective of this study was to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) for detection of PSV immunoglobulin G (IgG) antibody in pigs. A PSV strain, named SHPD202148, was first isolated from the fecal samples of piglets. Its structural protein, VP1, was prokaryotic-expressed in the pET expression system, followed by purification. Using the recombinant protein with reactogenicity as coating antigen, an i-ELISA, characterized by high sensitivity and specificity, had a detection limit at 1:12 800 dilution with a determined cutoff value of 0.352. Finally, field sera collected from different pig herds were tested in parallel by the serum neutralization (SN) test. The result showed that 126 samples were positive and 36 were negative, with an agreement of 97.0% in both cases. This i-ELISA can be used as an alternative serological test for detecting antibodies against PSV in blood serum.

Le sapelovirus porcin (PSV) est un entérovirus nouvellement émergent largement répandu en Chine. Puisqu'il n'y a pas de test sérologique clinique pour le PSV, l'objectif de cette étude était de développer un test immuno-enzymatique indirect (i-ELISA) pour la détection d'immunoglobuline G (IgG) anti-PSV chez les porcs. Une souche de PSV, nommée SHPD202148, a d'abord été isolée à partir d'échantillons fécaux de porcelets. Sa protéine structurale, VP1, a été exprimée par un procaryote dans le système d'expression pET, suivie d'une purification. Utilisant la protéine recombinante à réactogénicité comme antigène de revêtement, un i-ELISA, caractérisé par une sensibilité et une spécificité élevées, avait une limite de détection à une dilution de 1:12 800 avec une valeur seuil déterminée de 0,352. Enfin, des sérums de terrain collectés dans différents troupeaux de porcs ont été testés en parallèle par le test de neutralisation sérique (SN). Le résultat a montré que 126 échantillons étaient positifs et 36 étaient négatifs, avec un accord de 97,0 % dans les deux cas. Cet i-ELISA peut être utilisé comme test sérologique alternatif pour détecter les anticorps anti-PSV dans le sérum sanguin.(Traduit par Docteur Serge Messier).

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.

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.

Genomic Divergence Characterization and Quantitative Proteomics Exploration of Type 4 Porcine Astrovirus.

Porcine astrovirus (PAstV) has been identified as an important diarrheic pathogen with a broad global distribution. The PAstV is a potential pathogen to human beings and plays a role in public health. Until now, the divergence characteristics and pathogenesis of the PAstV are still not well known. In this study, the PAstV-4 strain PAstV/CH/2022/CM1 was isolated from the diarrheal feces of a piglet in Shanghai, which was identified to be a recombination of PAstV4/JPN (LC201612) and PAstV4/CHN (JX060808). A time tree based on the ORF2 protein of the astrovirus demonstrated that type 2-5 PAstV (PAstV-2 to 5) diverged from type 1 PAstV (PAstV-1) at a point from 1992 to 2000. To better understand the molecular basis of the virus, we sought to explore the host cell response to the PAstV/CH/2022/CM1 infection using proteomics. The results demonstrate that viral infection elicits global protein changes, and that the mitochondria seems to be a primary and an important target in viral infection. Importantly, there was crosstalk between autophagy and apoptosis, in which ATG7 might be the key mediator. In addition, the NOD-like receptor X1 (NLRX1) in the mitochondria was activated and participated in several important antiviral signaling pathways after the PAstV/CH/2022/CM1 infection, which was closely related to mitophagy. The NLRX1 may be a crucial protein for antagonizing a viral infection through autophagy, but this has yet to be validated. In conclusion, the data in this study provides more information for understanding the virus genomic characterization and the potential antiviral targets in a PAstV infection.

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.

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.

The Gut Viral Metagenome Analysis of Domestic Dogs Captures Snapshot of Viral Diversity and Potential Risk of Coronavirus.

The close relations between dogs (Canis lupus familiaris) and humans lay a foundation for cross species transmissions of viruses. The co-existence of multiplex viruses in the host accelerate viral variations. For effective prediction and prevention of potential epidemic or even pandemic, the metagenomics method was used to investigate the gut virome status of 45 domestic healthy dogs which have extensive contact with human beings. A total of 248.6 GB data (505, 203, 006 valid reads, 150 bp in length) were generated and 325, 339 contigs, which were best matched with viral genes, were assembled from 46, 832, 838 reads. In the aggregate, 9,834 contigs (3.02%) were confirmed for viruses. The top 30 contigs with the most reads abundance were mapped to DNA virus families Circoviridae, Parvoviridae and Herpesviridae; and RNA virus families Astroviridae, Coronaviridae and Picornaviridae, respectively. Numerous sequences were assigned to animal virus families of Astroviridae, Coronaviridae, Circoviridae, etc.; and phage families of Microviridae, Siphoviridae, Ackermannviridae, Podoviridae, Myoviridae and the unclassified phages. Further, several sequences were homologous with the insect and plant viruses, which reflects the diet and habitation of dogs. Significantly, canine coronavirus was uniquely identified in all the samples with high abundance, and the phylogenetic analysis therefore showed close relationship with the human coronavirus strain 229E and NL63, indicating the potential risk of canine coronavirus to infect humans by obtaining the ability of cross-species transmission. This study emphasizes the high detection frequency of virus harbored in the enteric tract of healthy contacted animal, and expands the knowledge of the viral diversity and the spectrum for further disease-association studies, which is meaningful for elucidating the epidemiological and biological role of companion animals in public health.

The Complex Co-infections of Multiple Porcine Diarrhea Viruses in Local Area Based on the Luminex xTAG Multiplex Detection Method.

The large-scale outbreaks of severe diarrhea caused by viruses have occurred in pigs since 2010, resulting in great damage to the pig industry. However, multiple infections have contributed to the outbreak of the disease and also resulted in great difficulties in diagnosis and control of the disease. Thus, a Luminex xTAG multiplex detection method, which was more sensitive and specific than general multiplex PCR method, was developed for the detection of 11 viral diarrhea pathogens, including PKoV, PAstV, PEDV, PSaV, PSV, PTV, PDCoV, TGEV, BVDV, PoRV, and PToV. To investigate the prevalence of diarrhea-associated viruses responsible for the outbreaks, a total of 753 porcine stool specimens collected from 9 pig farms in Shanghai during 2015-2018 were tested and the pathogen spectrums and co-infections were analyzed. As a result, PKoV, PAstV and PEDV were most commonly detected viruses in diarrheal pigs with the rate of 38.65% (291/753), 20.32% (153/753), and 15.54% (117/753), respectively. Furthermore, multiple infections were commonly seen, with positive rate of 28.42%. Infection pattern of the viral diarrhea pathogens in a specific farm was changing, and different farms had the various diarrhea infection patterns. A longitudinal investigation showed that PEDV was the key pathogen which was closely related to the death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease. Furthermore, the surveillance confirmed that variant enteropathogenic viruses were leading etiologic agents of porcine diarrhea, either mono-infection or co-infections of PKoV were common in pigs in Shanghai, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status, suggesting that controlling porcine diarrhea might be more complex than previously thought. The study provides a better understanding of diarrhea viruses in piglets, which will aid in better preventing and controlling epidemics of viral porcine diarrhea.

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.

Rhein protects the myocardiac cells against hypoxia/reoxygention-induced injury by suppressing GSK3ß activity.

BACKGROUND: Rhein, an anthraquinone compound isolated from rhubarb, has been shown to protect the pancreatic ß cells from hyperglycemia induced apoptosis in our previous studies. PURPOSE: In the present study, we examined whether rhein can protect myocardial cells against ischemia reperfusion (I/R)-induced apoptosis and investigated the underlying mechanism. METHODS: We used an in vitro model of myocardial hypoxia/reoxygenation (H/R) injury. H9c2 cells were incubated with rhein for 1 h and then subjected to hypoxia for 6 h, followed by reoxygenation for 2 h. Cells viability, apoptosis and ROS were assayed for the treated cells. AKT, p-AKT, GSK3ß, p- GSK3ß, P38 and p-P38 proteins were analyzed using Western blotting. PI3K/AKT inhibitor, LY294002, and GSK3ß siRNA were also used to determine the signaling pathways involved in the protection by rhein. RESULTS: Rhein increased viability, decreased apoptosis and ROS production, of the cells that were exposed to H/R. Rhein also increased the phosphorylation of AKT and GSK3ß, an effect that was eliminated by LY294002. GSK3ß silencing by siRNA showed similar effect as LY294002. The p-P38 level was upregulated by H/R and downregulated in the presence of rhein; however, the p-P38 downregulation was completely abolished by GSK3ß silencing. CONCLUSION: Rhein protects myocardial H9c2 cells against hypoxia/reoxygenation induced injury via AKT/ GSK3ß/p38 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.

Quantitative measurement of carbon isotopic composition in CO2 gas reservoir by Micro-Laser Raman spectroscopy.

The use of Micro-Laser Raman spectroscopy technology for quantitatively determining gas carbon isotope composition is presented. In this study, 12CO2 and 13CO2 were mixed with N2 at various molar fraction ratios to obtain Raman quantification factors (F12CO2 and F13CO2), which provide a theoretical basis for calculating the δ13C value. And the corresponding values were 0.523 (0

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.

[Clinical detection of seven porcine diarrhea-associated viruses and evolution analysis of porcine kobuvirus].

In this study, a multiplex RT-PCR method was developed for detection of seven diarrhea-associated porcine viruses, including porcine teschovirus (PTV), porcine sapovirus (PSV), porcine deltacornavirus (PDCoV), porcine kobuvirus (PKV), porcine sapovirus (PSaV), porcine astrovirus (PAstV) and porcine torovirus (PToV). A total of 419 samples were screened by this method and results showed that PKV had the highest positive rate of 26.98%?45.79% and its mixed infection rate reached 9.52%-18.54%. On account of high positive rate of PKV and its important role in diarrhea disease, complete genomic sequences of three PKV positive samples were further sequenced. Three PKV labeled as PD-PKV, JS-PKV and CM-PKV were classified into porcine kobuvirus genus and had far genetic distance with other kobuviruses. The complete genome homologies among them were 88.1%-89.1%. CM-PKV had the highest identity with the Chinese strain JS-02a-CHN/2013 reported in 2013 while JS-PKV and PD-PKV were most closed to the K-30-HUN/2008/HUN strain reported in Hungary in 2008. This illustrates the significant genetic differences of the different PKV isolates in Shanghai while its relationship with the viral pathogenicity still needs to be explored. This research provides references for further understanding the prevalence of PKV and its role in swine diarrhea.