IntroductionPyroptosis is a distinctive form of inflammatory cell death, mediated by the activation of the inflammasome, which initiates a potent inflammatory response. Long non-coding RNAs (lncRNAs) modulate pyroptosis by targeting microRNAs and their target genes through a competing endogenous RNA (ceRNA) mechanism. Our previous research has confirmed that Polysaccharide of Atractylodes macrocephala Koidz (PAMK) can alleviate inflammatory liver injury in mice caused by lipopolysaccharide (LPS), but the specific molecular mechanism remains unclear. Additionally, recent studies have identified recruited macrophages in the liver as a key component of both acute and chronic liver inflammation. This study aimed to explore the impact of PAMK on LPS-induced macrophage pyroptosis and its molecular mechanism in mitigating inflammatory liver injury in mice.MethodsC57BL/6 mice were subjected to LPS-induced liver injury with or without PAMK pretreatment. Histopathological analysis, qRT-PCR, Western blot, and ELISA were performed to assess liver damage and pyroptosis markers. In RAW264.7 macrophages, dual-luciferase assays validated ceRNA interactions, while gain/loss-of-function experiments elucidated molecular mechanisms.ResultsIn this study, we found that PAMK alleviated LPS-induced inflammatory liver injury in mice and modulates macrophage pyroptosis through the lncRNA GAS5/miR-223/NLRP3 axis.ConclusionWe conclude that there is a ceRNA relationship between GAS5, miR-223-3p, and NLRP3; PAMK alleviates LPS-induced pyroptosis in macrophages through the lncRNA GAS5/miR-223-3p/NLRP3 axis; and PAMK intervention in the macrophage pyroptosis process subsequently alleviates liver inflammation.

This study explores the conditions and circumstances of officials being held strictly accountable. Using fuzzy set qualitative comparative analysis (fs/QCA), we study accountability of officials in 42 emergency incidents in China. The analysis reveals different but functionally equivalent causal conditions leading to strict accountability of officials. The findings highlight the importance of responsibility type and accident severity for strict accountability and demonstrate its mechanism and causal relationships. The study's contribution provides empirical evidence of officials' strict accountability in a non-Western context, revealing its antecedents and causal chains and offering insights to policymakers for strengthening the accountability framework.

Lactate dehydrogenase A (LDHA) plays a crucial role in regulating lactate synthesis in various biological processes. Lactate, a byproduct of glycometabolism, has been recognized as a unique molecule with implications in both metabolism and immunity. Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is a highly contagious and severe infectious disease that primarily affects pigs. Prior research has shown that CSFV infection disrupts the normal glycolytic process, leading to an accumulation of lactate within the host. Nevertheless, it remains unclear whether there is mutual regulation between the CSFV and LDHA-lactate axis. Here, we have found that CSFV infection increases LDHA expression in vivo and in vitro, which may be attributed to attenuated ISGylation of LDHA. Furthermore, CSFV infection induces L-lactate production via LDHA dependence in vitro. The cellular biology research on LDHA has revealed that LDHA not only localizes to the mitochondria but also inhibits PINK1-Parkin-mediated mitophagy. Through various experimental techniques such as western blot to detect mitophagy marker proteins, laser confocal microscopy to observe the flow of mitophagy, and transmission electron microscopy to assess changes in the number of mitochondria enclosed within autophagosome-like vesicles, it has been discovered that the addition of exogenous lactate can inhibit PINK1-Parkin-mediated mitophagy. Importantly, we have observed that lactate activates the JAK1-STAT1-ISG15 network and suppresses CSFV replication by antagonizing CCCP-induced mitophagy. These results represent the first report on the mechanisms through which the LDHA-lactate axis regulates mitophagy, the JAK-STAT pathway, and CSFV replication. This study provides novel insights into the roles of the LDHA-lactate axis in glycometabolism and viral replication. IMPORTANCE:This research unveils how CSFV interacts with cellular metabolism through LDHA. By revealing LDHA's dual role and how lactate influences cellular processes during CSFV infection, this study uncovers new pathways for viral replication. These findings not only deepen our understanding of viral-host interactions but also open doors for innovative antiviral strategies centered around manipulating cellular metabolism.

Non-alcoholic fatty liver disease (NAFLD) is increasingly prevalent, but treatment options are limited. Previous studies have demonstrated the hepatoprotective effects of Polysaccharide of Atractylodes macrocephala Koidz (PAMK) against liver injury induced by various causes, but its potential in alleviating NAFLD remains unknown. This study aimed to investigate the potential of PAMK in improving NAFLD and its regulatory effects on gene transcription during the process. The results indicated that Highfatdiet (HFD) could induce NAFLD in mice, and PAMK was found to alleviate symptoms of NAFLD and mitigate liver injury caused by HFD. Transcriptome analysis revealed that PAMK affects both metabolic and inflammatory pathways, suggesting its dual impact on maintaining metabolic homeostasis and suppressing inflammation during NAFLD progression. The results of liver biochemical markers, key genes of the cholesterol pathway, glucose (GLU), and inflammatory factors further demonstrated the dual role of PAMK in metabolism and inflammation.

Non-alcoholic fatty liver disease (NAFLD) not only could cause abnormal lipid metabolism in the liver, but also could cause liver inflammation. Previous studies have shown that Polysaccharide of Atractylodes macrocephala Koidz (PAMK) could alleviate animal liver inflammatory damage and alleviate NAFLD in mice caused by high-fat diet(HFD), but regulation of liver inflammation caused by NAFLD has rarely been reported. In this study, an animal model of non-alcoholic fatty liver inflammation in the liver of mice was established to explore the protective effect of PAMK on the liver of mice. The results showed that PAMK could alleviate the abnormal increase of body weight and liver weight of mice caused by HFD, alleviate the abnormal liver structure of mice, reduce the level of oxidative stress and cytokine secretion in the liver of mice, and downregulate the mRNA expression of TLR4, MyD88, NF-κB and protein expression of P-IκB, P-NF-κB-P65, TLR4, MyD88, NF-κB in the liver. These results indicate that PAMK could alleviate hepatocyte fatty degeneration and damage, oxidative stress and inflammatory response of the liver caused by NAFLD in mice.

Ferroptosis is a distinctive form of iron-dependent cell death characterized by significant ultrastructural changes in mitochondria. Given the crucial involvement of mitochondria in various cellular processes such as reactive oxygen species production, energy metabolism, redox status, and iron metabolism, mounting evidence suggests a vital role of mitochondria in the regulation and execution of ferroptosis. Furthermore, there exists a strong correlation between ferroptosis and various diseases. In this review, we aim to summarize the mechanisms underlying the induction and defense of ferroptosis, emphasizing the influence of mitochondria on this intricate process. Additionally, we provide an overview of the role of ferroptosis in disease, particularly cancer, and elucidate the mechanisms by which drugs targeting mitochondria impact ferroptosis. By presenting a theoretical foundation and reference point, this review aims to contribute to both basic cell biology research and the investigation of clinically relevant diseases.

CSFV (classical swine fever virus) is currently endemic in developing countries in Asia and has recently re-emerged in Japan. Under the pressure of natural selection pressure, CSFV keeps evolving to maintain its ecological niche in nature. CSFV has evolved mechanisms that induce immune depression, but its pathogenic mechanism is still unclear. In this study, using transcriptomics and metabolomics methods, we found that CSFV infection alters innate host immunity by activating the interferon pathway, inhibiting host inflammation, apoptosis, and remodelling host metabolism in porcine alveolar macrophages. Moreover, we revealed that autophagy could alter innate immunity and metabolism induced by CSFV infection. Enhanced autophagy further inhibited CSFV-induced RIG-I-IRF3 signal transduction axis and JAK-STAT signalling pathway and blocked type I interferon production while reducing autophagy inhibition of the NF-κB signalling pathway and apoptosis in CSFV infection cells. Furthermore, the level of CSFV infection-induced glycolysis and the content of lactate and pyruvate, as well as 3-phosphoglyceraldehyde, a derivative of glycolysis converted to serine, was altered by autophagy. We also found that silencing HK2 (hexokinase 2), the rate-limiting enzyme of glycolytic metabolism, could induce autophagy but reduce the interferon signalling pathway, NF-κB signalling pathway, and inhibition of apoptosis induced by CSFV infection. In addition, inhibited cellular autophagy by silencing ATG5 or using 3-Methyladenine, could backfill the inhibitory effect of silencing HK2 on the cellular interferon signalling pathway, NF-κB signalling pathway, and apoptosis.

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host’s immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-β promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2′-5′oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1β. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-β, and IL-1β. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies.

Mitochondria are important organelles involved in cell metabolism and programmed cell death in eukaryotic cells and are closely related to the innate immunity of host cells against viruses. Mitophagy is a process in which phagosomes selectively phagocytize damaged or dysfunctional mitochondria to form autophagosomes and is degraded by lysosomes, which control mitochondrial mass and maintain mitochondrial dynamics and cellular homeostasis. Innate immunity is an important part of the immune system and plays a vital role in eliminating viruses. Viral infection causes many physiological and pathological alterations in host cells, including mitophagy and innate immune pathways. Accumulating evidence suggests that some virus promote self-replication through regulating mitophagy-mediated innate immunity. Clarifying the regulatory relationships among mitochondria, mitophagy, innate immunity, and viral infection will shed new insight for pathogenic mechanisms and antiviral strategies. This review systemically summarizes the activation pathways of mitophagy and the relationship between mitochondria and innate immune signaling pathways, and then discusses the mechanisms of viruses on mitophagy and innate immunity and how viruses promote self-replication by regulating mitophagy-mediated innate immunity.

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I–mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

Canine influenza (CI) is a contagious respiratory disease in dogs, which poses a threat to canine health. A safe, high-yield vaccine seed virus is critical for CI vaccine development. We developed a PR8-based reassortant H3N2 canine influenza virus (RT CIV) using the reverse genetic method and evaluated its yield in canine kidney epithelial (MDCK) cells, Vero cells, and specific pathogen-free (SPF) chicken embryos. Mice and dogs were infected with RT CIV, and the pathogenicity was evaluated. The viral titers of RT CIV increased in MDCK cells, Vero cells, and SPF chicken embryos; the HA yield in SPF chicken embryos increased 4-fold. However, RT CIV was not lethal to mice, and it showed similar virulence as wild-type CIV. RT CIV also showed minimal pathogenicity in dogs, which manifested as mild fever and rhinorrhea for the first two days post-infection. Thus, RT CIV carrying the internal gene cassette from PR8 showed almost no pathogenicity in dogs. And the reassortant virus inactivated vaccine could provide complete protection against H3N2 CIV. To our knowledge, this is the first report on the pathogenicity of PR8-based reassortant H3N2 CIV in dogs. These studies are relevant for developing a high-yield and safe CI vaccine.

Avian-origin H3N2 canine influenza viruses (CIVs) cause severe contagious respiratory disease in dogs, and quickly adapt to new environments. To further understand the mechanism of virus infection and host-virus interactions, we characterized the complete phosphoproteome of dogs infected with H3N2 CIV. Nine-week-old Beagle dogs were inoculated intranasally with 10(6) EID50 of A/canine/Guangdong/04/2014 (H3N2) virus. Lung sections were harvested at 5 days post-inoculation (dpi) and processed for global and quantitative analysis of differentially expressed phosphoproteins. A total of 1,235 differentially expressed phosphorylated proteins were identified in the dog lung after H3N2 CIV infection, and 3,016 modification sites were identified among all differentially expressed proteins. We then performed an enrichment analysis of functional annotations using Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) database analyses to predict the functions of the identified differential phosphoproteins. Our data indicate that H3N2 CIV infection causes dramatic changes in the host protein phosphorylation of dog lungs. To our knowledge, this is the first study to assess the effect of H3N2 CIV infection on the phosphoproteome of beagles. These data provide novel insights into H3N2-CIV-triggered regulatory phosphorylation circuits and signaling networks and may improve our understanding of the mechanisms underlying CIV pathogenesis in dogs.

MDA5 belongs to the RIG-I-like receptor family, which is involved in innate immunity. During viral infection, MDA5 generates an antiviral response by recognizing the ligand to activate interferon. However, the role and mechanism of MDA5 in canine influenza virus (CIV) infection are unclear. To understand the mechanism of canine MDA5-mediated innate immunity during CIV infection, we detected the distribution of MDA5 in beagles, and the structural prediction showed that MDA5 was mainly composed of a CARD domain, RD domain, and DExD/H helix structure. Moreover, we found that MDA5 inhibits CIV replication. Furthermore, in the dual luciferase assay, we revealed that the CARD region of MDA5 strongly activated the IFN-β promoter and mainly transmitted signals through the CARD region. Overexpression of the CARD region of MDA5 revealed that the MDA5-mediated signaling pathway could transmit signals by activating the IRF3/NF-κB and IRF3 promoters, promoting the expression of antiviral proteins and cytokine release, thereby inhibiting CIV replication. Upon silencing of MDA5, cytokine production decreased, while the replication ability of CIV was increased. Thus, this study revealed a novel mechanism by which MDA5 mediated CIV infection and provided new avenues for the development of antiviral strategies.

The canine influenza virus (CIV) outbreaks have raised concerns as they pose a threat to the health of dogs. The successful construction of a canine influenza (CI) infection model is essential to study the CIV. Here we investigated the pathogenicity of different infectious doses of H3N2 CIV in Beagle dogs. Thirty-seven healthy Beagle dogs were used in the experiment and were infected with 10(3), 10(4), 10(5), and 10(6) 50% egg-infectious doses (EID50). Compared to the dogs in the other three groups, those in the 10(6) EID50 group presented with obvious clinical symptoms, high virus titer, and typical pathological changes. Considering the ensemble of clinical scores, body temperature, virus shedding, lung lesions, pathological section scores, and visceral virus titers, we determined that 10(6) EID50 is the minimum infectious dose for the Beagle infection model. The other three infectious doses had almost no clinical symptoms. These results indicate that 10(6) EID50 is the minimum infectious dose of H3N2 CIV that can cause obvious clinical manifestations and pathological changes associated with CI in Beagle dogs. The theoretical framework developed in this research will guide the establishment of an infection model of CIV for future research.

Currently, Canine influenza virus (CIV) H3N2 is continuously circulating in dog populations in China, Korea, and the United States (US). Both influenza SA-α-2,3-Gal and SA-α-2,6-Gal receptors have been observed in the respiratory tracts of both horses and dogs. Hence, the increasing number of CIV H3N2 cases in the world indicates a potential risk for transspecies transmission to horses with dog exposure. Here, a seroepidemiological survey of CIV H3N2 infections in horses was conducted using hemagglutination inhibition (HI), microneutralization (MN) and the chicken embryo neutralization test (CENT). From April 2014 to November 2016, 399 sera from race horses were collected in Guangzhou, Dongguan, Huizhou, and Shenzhen in China. Nine specimens (2.2%, 9/399) were positive for CIV H3N2 with HI titers ≥ 1:20, MN titers ≥ 1:80 and CENT titers ≥ 1:80. Furthermore, these positive horses showed significant correlation with dog exposure, and some dogs (20%, 3/15) from the same riding clubs as the positive horses also possessed antibodies against CIV H3N2. This study is the first to provide seroepidemiological evidence of CIV H3N2 infection in horses with exposure to dogs. Based on these findings, continuous serological and virological surveillance of CIV H3N2 infection among horses is imperative, and further animal experiments should be performed.

Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID50) of 106, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA-mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA-mRNA pairs, respectively. One hundred and twenty negative miRNA-mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection.

As important companion animals, dogs may serve as intermediate hosts for transmitting influenza virus to humans. However, knowledge regarding H3N2 canine influenza virus (CIV) pathogenicity is not comprehensive, which directly affects the animal models of pathogenicity in H3N2 CIV vaccine research. Here, to assess H3N2 CIV pathogenicity, we utilized 30 ten-week-old purpose-bred beagles intratracheally or intranasally inoculated with 106 50% egg-infectious dose. Intratracheal inoculation was more virulent to dogs than intranasal inoculation as shown by lung pathology score, histopathological changes, clinical symptoms, and body temperature. More intense virus replication was observed in the upper and lower respiratory tracts by intratracheal than intranasal inoculation according to nasal swabs, various organ virus titers, and antigen expression. These results may enhance the H3N2 CIV infection model, providing a more complete experimental basis for studying intrinsic H3N2 CIV pathogenic mechanism, and also serving a reference role for CIV prevention and treatment.

Equine pegivirus (EPgV) is a newly discovered equine virus, which is taxonomically classified in the Pegivirus genus of the Flaviviridae family. Until now, only the complete genome sequence of the first reported EPgV strain, from the USA (strain name: C0035) is available on online databases. Considering this, horse serum samples were collected from horses in China and screened for EPgV RNA by RT-PCR. One EPgV strain, LW/2016, was obtained and its near-complete genome sequence was acquired by standard PCR. Further analysis of its nucleotide sequence indicates LW/2016 is genetically divergent from C0035, with a nucleotide identity of 89.02%. These two viruses clustered into two independent branches following phylogenetic analysis of on the NS3 and NS5B genes. To our knowledge, this is the first report of genetic divergence in the EPgV genome.

Objective] To study the natural recombination, cross host transmission and prevalence of feline panleukopenia virus( FPLV) in Guangzhou area.[Method]Cells were isolated from stool samples of cats seemingly infected with FPLV, genome DNAs were extracted from positive samples, and complete virus genome sequences were amplified by PCR and were compared with relevant reference sequences from GenBank using genetic evolution analysis. The major amino acid sites were analyzed for differences between VP2 gene and NS1 gene. [Result]Two FPLV strains were successfully isolated and the complete genome sequences of FPLV were obtained. The genetic evolution analysis indicated that FPLV-GZ01 and FPLV-GZ02 which were isolated from Guangzhou area belonged to the same branch with the other isolated strains FPLV-XJ1 and FPLV-HRB, and implied FPLV-GZ01 and FPLV-GZ02 evolved from FPLV-XJ01. The NS1 gene phylogenetic analysis demonstrated that FPLV might have recombined with CPV-447 . Analysis of major amino acid sites indicated that FPLV was more conservative than CPV in the genetic variation of the VP2 gene. FPLV-GZ01 and FPLV-GZ02 had different degrees of mutation in the amino acid sites of the NS1 gene. [Conclusion]The feline panleukopenia viruses isolated from Guangzhou area have been still recombining and evolving.

Equine hepacivirus (EqHV), equine pegivirus (EPgV) and Theiler's disease-associated virus (TDAV) are three novel equine viruses in the family Flaviviridae. EqHV and EPgV have been identified to circulate in the equine population worldwide, whereas TDAV has not been detected in equines since the first reported case. To date, no studies have focused on investigating EqHV, EPgV and TDAV in commercial equine sera or equine blood products in China. Considering the potential threat of EqHV, EPgV and TDAV to biosecurity and considering their possible influences on research results, equine sera for cell culture propagation and pregnant mare serum gonadotropin (PMSG) were purchased from different companies in China and investigated for EqHV, EPgV and TDAV in this study. By performing nested PCR or two rounds of PCR targeting the viral NS3 gene, four serum samples were confirmed to be EqHV-, EPgV-, or TDAV-RNA positive; all of the PMSG samples were negative for these three viruses. Subsequent sequencing results indicated that the serum samples contained multiple viral variants of EqHV, EPgV or TDAV, and a genetic analysis based on the partial NS3 gene of the three equine viruses was performed. Our study is the first to confirm the presence of EqHV, EPgV and TDAV in equine sera for cell culture propagation that is commercially available in China and provides the first demonstration of the presence of TDAV in China.