Fast detection of hypobromous acid in cells and the water environment using a lysosome-targeted fluorescent probe.

A new fluorescent probe SWJT-23 with lysosomal targeting ability for detection of hypobromous acid (HBrO) was synthesised based on the naphthalimide skeleton. This probe exhibited a fast response (within 3s), a low detection limit (1.24 nM), excellent selectivity and a high fluorescence quantum yield (Φ = 0.490). Moreover, SWJT-23 not only realized the sensitive detection of HBrO in cells and water samples, but also was fabricated as a paper-based sensor. In consequence, SWJT-23 is expected to be an efficient and powerful tool for monitoring HBrO in organisms and the environment in realistic scenarios.

Seneca Valley virus 3C protease cleaves OPTN (optineurin) to Impair selective autophagy and type I interferon signaling.

Seneca Valley virus (SVV) causes vesicular disease in pigs, posing a threat to global pork production. OPTN (optineurin) is a macroautophagy/autophagy receptor that restricts microbial propagation by targeting specific viral or bacterial proteins for degradation. OPTN is degraded and cleaved at glutamine 513 following SVV infection via the activity of viral 3C protease (3C[pro]), resulting in N-terminal and a C-terminal OPTN fragments. Moreover, OPTN interacts with VP1 and targets VP1 for degradation to inhibit viral replication. The N-terminal cleaved OPTN sustained its interaction with VP1, whereas the degradation capacity targeting VP1 decreased. The inhibitory effect of N-terminal OPTN against SVV infection was significantly reduced, C-terminal OPTN failed to inhibit viral replication, and degradation of VP1 was blocked. The knockdown of OPTN resulted in reduced TBK1 activation and phosphorylation of IRF3, whereas overexpression of OPTN led to increased TBK1-IRF3 signaling. Additionally, the N-terminal OPTN diminished the activation of the type I IFN (interferon) pathway. These results show that SVV 3C[pro] targets OPTN because its cleavage impairs its function in selective autophagy and type I IFN production, revealing a novel model in which the virus develops diverse strategies for evading host autophagic machinery and type I IFN response for survival.Abbreviations: Co-IP: co-immunoprecipitation; GFP-green fluorescent protein; hpi: hours post-infection; HRP: horseradish peroxidase; IFN: interferon; IFNB/IFN-ß: interferon beta; IRF3: interferon regulatory factor 3; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; OPTN: optineurin; PBS: phosphate-buffered saline; SVV: Seneca Valley virus; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; UBAN: ubiquitin binding in TNIP/ABIN (TNFAIP3/A20 and inhibitor of NFKB/NF-kB) and IKBKG/NEMO; UBD: ubiquitin-binding domain; ZnF: zinc finger.

Seneca Valley virus 3Cpro antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals.

IMPORTANCE: Type I interferon (IFN) signaling plays a principal role in host innate immune responses against invading viruses. Viruses have evolved diverse mechanisms that target the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway to modulate IFN response negatively. Seneca Valley virus (SVV), an emerging porcine picornavirus, has received great interest recently because it poses a great threat to the global pork industry. However, the molecular mechanism by which SVV evades host innate immunity remains incompletely clear. Our results revealed that SVV proteinase (3Cpro) antagonizes IFN signaling by degrading STAT1, STAT2, and IRF9, and cleaving STAT2 to escape host immunity. SVV 3Cpro also degrades karyopherin 1 to block IFN-stimulated gene factor 3 nuclear translocation. Our results reveal a novel molecular mechanism by which SVV 3Cpro antagonizes the type I IFN response pathway by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which has important implications for our understanding of SVV-evaded host innate immune responses.

Impact of influenza virus infection on lung microbiome in adults with severe pneumonia.

BACKGROUND: Bacterial and viral infections are commonly implicated in the development of pneumonia. We aimed to compare the diversity and composition of lung bacteria among severe pneumonia patients who were influenza virus positive (IFVP) and influenza virus negative (IFVN). METHODS: Bronchoalveolar lavage fluid specimens were procured from patients diagnosed with severe pneumonia to investigate the microbiome utilizing 16S-rDNA sequencing. The alpha diversity of the microbiome was evaluated employing Chao1, Shannon, and Simpson indexes, while the beta diversity was assessed using principal component analysis and principal coordinate analysis. Linear discriminant analysis effect size (LEfSe) was employed to determine the taxonomic differences between the IFVP and IFVN groups. RESULTS: A total of 84 patients with 42 in the IFVP group and 42 in the IFVN group were enrolled. Slightly higher indexes of Shannon and Simpson were observed in the IFVP group without statistically significant difference. The dominant bacterial genera were Streptococcus, Klebsiella, Escherichia-Shigella in the IFVN group and Acinetobacter, Streptococcus, Staphylococcus in the IFVP group. Streptococcus pneumoniae and Acinetobacter baumannii were the most abundant species in the IFVN and IFVP groups, respectively. LEfSe analysis indicated a greater abundance of Klebsiella in the IFVN group. CONCLUSIONS: Individuals with severe pneumonia infected with IFV exhibit heightened susceptibility to certain bacteria, especially Acinetobacter baumannii, and the underlying mechanism of the interaction between IFV and Acinetobacter baumannii in the progression of pneumonia needs further investigation.

A fluorescein-based fluorescent probe for fast detection of malondialdehyde and its imaging study.

A simple fluorescein derivative as a fluorescent probe was synthesized for the detection of malondialdehyde (MDA) through a synergistic reaction to achieve ring-opening of fluorescein and formation of a benzohydrazide derivative. It exhibited high sensitivity and selectivity for MDA detection. The probe could also detect MDA quickly (within 60 s) and visually via UV-vis and fluorescent modes. Moreover, this probe showed good performance in the imaging of MDA in living cells and bacteria.

Discriminatively Unsupervised Learning Person Re-Identification via Considering Complicated Images.

State-of-the-art purely unsupervised learning person re-ID methods first cluster all the images into multiple clusters and assign each clustered image a pseudo label based on the cluster result. Then, they construct a memory dictionary that stores all the clustered images, and subsequently train the feature extraction network based on this dictionary. All these methods directly discard the unclustered outliers in the clustering process and train the network only based on the clustered images. The unclustered outliers are complicated images containing different clothes and poses, with low resolution, severe occlusion, and so on, which are common in real-world applications. Therefore, models trained only on clustered images will be less robust and unable to handle complicated images. We construct a memory dictionary that considers complicated images consisting of both clustered and unclustered images, and design a corresponding contrastive loss by considering both kinds of images. The experimental results show that our memory dictionary that considers complicated images and contrastive loss can improve the person re-ID performance, which demonstrates the effectiveness of considering unclustered complicated images in unsupervised person re-ID.

Characterization of avain metapneumovirus subgroup C isolated from chickens in Beijing, China.

Avian metapneumovirus (aMPV) is an important causative agent that causes acute respiratory disease and egg-dropping in chickens and turkeys. Here, we characterized an aMPV subgroup C (aMPV/C) from 320-day-old broiler breeder chickens with severe respiratory diseases in Beijing, China, as evidenced by RT-PCR typing and confirmation of the nucleoprotein (N) gene sequence. The N gene sequence of the aMPV/C strain (designated BJ17) exhibited no deletions or insertions and possessed 94.6% to 99.6% identity to those of published aMPV/C isolates. The phylogenetic tree of the nucleotide sequences constructed using the neighbor-joining clustering method showed that the BJ17 strain formed one cluster with other aMPV/C viruses and formed one subcluster with published Chinese aMPV/C isolates regardless of Muscovy duck or chicken origins. Comparative analysis of the N proteins showed that a unique amino acid residue D at position 110 might be associated with regional distribution due to its occurrence in all the Chinese aMPV/C isolates only. Strain BJ17 was successfully isolated by cultured Vero cell passage and further inoculated in 3-wk-old specific-pathogen-free chickens for the examination of pathogenicity. Animal experimental results showed that BJ17-inoculated chickens had severe respiratory diseases and inflammatory lesions, as demonstrated by pathological changes and aMPV antigen in the nasal turbinate, tracheae, and lung tissues. These results enrich the available information regarding the epidemiology and pathogenicity of aMPV/C in chickens, which may facilitate the development of effective measures against aMPV/C infection in China.

Effects of OxyR regulator on oxidative stress, Apx toxin secretion and virulence of Actinobacillus pleuropneumoniae.

Introduction: Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, poses a significant threat to global swine populations due to its high prevalence, mortality rates, and substantial economic ramifications. Understanding the pathogen's defense mechanisms against host-produced reactive oxygen species is crucial for its survival, with OxyR, a conserved bacterial transcription factor, being pivotal in oxidative stress response. Methods: This study investigated the presence and role of OxyR in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis was conducted on an oxyR disruption mutant to delineate the biological activities influenced by OxyR. Additionally, specific assays were employed to assess urease activity, catalase expression, ApxI toxin secretion, as well as adhesion and invasion abilities of the oxyR disruption mutant on porcine 3D4/21 and PT cells. A mice challenge experiment was also conducted to evaluate the impact of oxyR inactivation on A. pleuropneumoniae virulence. Results: OxyR was identified as a conserved regulator present in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis revealed the involvement of OxyR in multiple biological activities. The oxyR disruption resulted in decreased urease activity, elevated catalase expression, enhanced ApxI toxin secretion-attributed to OxyR binding to the apxIBD promoter-and reduced adhesion and invasion abilities on porcine cells. Furthermore, inactivation of oxyR reduced the virulence of A. pleuropneumoniae in a mice challenge experiment. Discussion: The findings highlight the pivotal role of OxyR in influencing the virulence mechanisms of A. pleuropneumoniae. The observed effects on various biological activities underscore OxyR as an essential factor contributing to the pathogenicity of this bacterium.

Seneca Valley Virus 3C pro Cleaves Heterogeneous Nuclear Ribonucleoprotein K to Facilitate Viral Replication.

Seneca Valley virus (SVV) has emerged as an important pathogen that is associated with idiopathic vesicular infection in pigs, causing a potential threat to the global swine industry. The heterogeneous nuclear ribonucleoprotein K (hnRNP K) that shuttles between the nucleus and cytoplasm plays an important role in viral infection. In this study, we observed that infection with SVV induced cleavage, degradation, and cytoplasmic redistribution of hnRNP K in cultured cells, which was dependent on the activity of viral 3Cpro protease. Also, the 3Cpro induced degradation of hnRNP K via the caspase pathway. Further studies demonstrated that SVV 3Cpro cleaved hnRNP K at residue Q364, and the expression of the cleavage fragment hnRNP K (aa.365-464) facilitates viral replication, which is similar to full-length hnRNP K, whereas hnRNP K (aa.1-364) inhibits viral replication. Additionally, hnRNP K interacts with the viral 5' untranslated region (UTR), and small interfering RNA (siRNA)-mediated knockdown of hnRNP K results in significant inhibition of SVV replication. Overall, our results demonstrated that the hnRNP K positively regulates SVV replication in a protease activity-dependent fashion in which the cleaved C-terminal contributes crucially to the upregulation of SVV replication. This finding of the role of hnRNP K in promoting SVV propagation provides a novel antiviral strategy to utilize hnRNP K as a potential target for therapy.

Seneca Valley Virus 3Cpro Mediates Cleavage and Redistribution of Nucleolin To Facilitate Viral Replication.

Seneca Valley virus (SVV) is a recently discovered pathogen that poses a significant threat to the global pig industry. It has been shown that many viruses are reliant on nucleocytoplasmic trafficking of nucleolin (NCL) for their own replication. Here, we demonstrate that NCL, a critical protein component of the nucleolus, is cleaved and translocated out of the nucleoli following SVV infection. Furthermore, our data suggest that SVV 3C protease (3Cpro) is responsible for this cleavage and subsequent delocalization from the nucleoli, and that inactivation of this protease activity abolished this cleavage and translocation. SVV 3Cpro cleaved NCL at residue Q545, and the cleavage fragment (aa 1 to 545) facilitated viral replication, which was similar to the activities described for full-length NCL. Small interfering RNA-mediated knockdown indicated that NCL is required for efficient viral replication and viral protein expression. In contrast, lentivirus-mediated overexpression of NCL significantly enhanced viral replication. Taken together, these results indicate that SVV 3Cpro targets NCL for its cleavage and redistribution, which contributes to efficient viral replication, thereby emphasizing the potential target of antiviral strategies for the control of SVV infection. IMPORTANCE The nucleolus is a subnuclear cellular compartment, and nucleolin (NCL) resides predominantly in the nucleolus. NCL participates in viral replication, translation, internalization, and also serves as a receptor for virus entry. The interaction between NCL and SVV is still unknown. Here, we demonstrate that SVV 3Cpro targets NCL for its cleavage and nucleocytoplasmic transportation, which contributes to efficient viral replication. Our results reveal novel function of SVV 3Cpro and provide further insight into the mechanisms by which SVV utilizes nucleoli for efficient replication.

Synergetic Contributions of Viral VP1, VP3, and 3C to Activation of the AKT-AMPK-MAPK-MTOR Signaling Pathway for Seneca Valley Virus-Induced Autophagy.

Seneca Valley virus (SVV), a member of the Picornaviridae family, can activate autophagy via the PERK and ATF6 unfolded protein response pathways and facilitate viral replication; however, the precise molecular mechanism that regulates SVV-induced autophagy remains unclear. Here, we revealed that SVV infection inhibited the phosphorylation of mechanistic target of rapamycin kinase (MTOR) and activated phosphorylation of the serine/threonine kinase AKT. We observed that activating AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), and p38 MAPK signaling by SVV infection promoted autophagy induction and viral replication; additionally, the SVV-induced autophagy was independent of the ULK1 complex. We further evaluated the role of viral protein(s) in the AKT-AMPK-MAPK-MTOR pathway during SVV-induced autophagy and found that VP1 induced autophagy, as evidenced by puncta colocalization with microtubule-associated protein 1 light chain 3 (LC3) in the cytoplasm and enhanced LC3-II levels. This might be associated with the interaction of VP1 with sequestosome 1 and promoting its degradation. In addition, the expression of VP1 enhanced AKT phosphorylation and AMPK phosphorylation, while MTOR phosphorylation was inhibited. These results indicate that VP1 induces autophagy by the AKT-AMPK-MTOR pathway. Additionally, expression of VP3 and 3C was found to activate autophagy induction via the ERK1/2 MAPK-MTOR and p38 MAPK-MTOR pathway. Taken together, our data suggest that SVV-induced autophagy has finely tuned molecular mechanisms in which VP1, VP3, and 3C contribute synergistically to the AKT-AMPK-MAPK-MTOR pathway. IMPORTANCE Autophagy is an essential cellular catabolic process to sustain normal physiological processes that are modulated by a variety of signaling pathways. Invading virus is a stimulus to induce autophagy that regulates viral replication. It has been demonstrated that Seneca Valley virus (SVV) induced autophagy via the PERK and ATF6 unfolded protein response pathways. However, the precise signaling pathway involved in autophagy is still poorly understood. In this study, our results demonstrated that viral proteins VP1, VP3, and 3C contribute synergistically to activation of the AKT-AMPK-MAPK-MTOR signaling pathway for SVV-induced autophagy. These findings reveal systemically the finely tuned molecular mechanism of SVV-induced autophagy, thereby facilitating deeper insight into the development of potential control strategies against SVV infection.

Seneca Valley virus 3Cpro degrades heterogeneous nuclear ribonucleoprotein A1 to facilitate viral replication.

Seneca Valley virus (SVV) is a recently-identified important pathogen that is closely related to idiopathic vesicular disease in swine. Infection of SVV has been shown to induce a variety of cellular factors and their activations are essential for viral replication, but whether heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) involved in SVV replication is unknown. The cytoplasmic redistribution of hnRNP A1 is considered to play an important role in the virus life cycle. Here, we demonstrated that SVV infection can promote redistribution of the nucleocytoplasmic shuttling RNA-binding protein hnRNP A1 to the cytoplasm from the nucleus, whereas hnRNP A1 remained mainly in the nucleus of mock-infected cells. siRNA-mediated knockdown of the gene encoding hnRNP A1 attenuated viral replication as evidenced by decreased viral protein expression and virus production, whereas its overexpression enhanced replication. Moreover, infection with SVV induced the degradation of hnRNP A1, and viral 3 C protease (3 Cpro) was found to be responsible for its degradation and translocation. Further studies demonstrated that 3 Cpro induced hnRNP A1 degradation through its protease activity, via the proteasome pathway. This degradation could be attenuated by a proteasome inhibitor (MG132) and inactivation of the conserved catalytic box in 3 Cpro. Taken together, these results presented here reveal that SVV 3 C protease targets cellular hnRNP A1 for its degradation and translocation, which is utilized by SVV to aid viral replication, thereby highlighting the control potential of strategies for infection of SVV.

Avian Metapneumovirus Subgroup C Induces Mitochondrial Antiviral Signaling Protein Degradation through the Ubiquitin-Proteasome Pathway.

The mitochondrial antiviral signaling (MAVS) protein, a critical adapter, links the upstream recognition of viral RNA to downstream antiviral signal transduction. However, the interaction mechanism between avian metapneumovirus subgroup C (aMPV/C) infection and MAVS remains unclear. Here, we confirmed that aMPV/C infection induced a reduction in MAVS expression in Vero cells in a dose-dependent manner, and active aMPV/C replication was required for MAVS decrease. We also found that the reduction in MAVS occurred at the post-translational level rather than at the transcriptional level. Different inhibitors were used to examine the effect of proteasome or autophagy on the regulation of MAVS. Treatment with a proteasome inhibitor MG132 effectively blocked MAVS degradation. Moreover, we demonstrated that MAVS mainly underwent K48-linked ubiquitination in the presence of MG132 in aMPV/C-infected cells, with amino acids 363, 462, and 501 of MAVS being pivotal sites in the formation of polyubiquitin chains. Finally, E3 ubiquitin ligases for MAVS degradation were screened and identified and RNF5 targeting MAVS at Lysine 363 and 462 was shown to involve in MAVS degradation in aMPV/C-infected Vero cells. Overall, these results reveal the molecular mechanism underlying aMPV/C infection-induced MAVS degradation by the ubiquitin-proteasome pathway.

Age-dependence of hypervirulent fowl adenovirus type 4 pathogenicity in specific-pathogen-free chickens.

Hypervirulent fowl adenovirus serotype 4 (hvFAdV-4) has emerged as a major pathogen of hepatitis-hydropericardium syndrome (HHS) with increased mortality in chickens, resulting in economic losses to the Chinese poultry industry since June 2015. Here, we isolated a hypervirulent FAdV-4 (hvFAdV-4) strain (designated GD616) from 25-day-old meat-type chickens with severe HHS in Guangdong Province China in June 2017. The whole genome of the strain GD616 shares high homology with those in the recently-reported hvFAdV-4 isolates in China, with natural deletions of ORF19 and ORF27. A comparative analysis of Hexon and Fiber-2 proteins revealed that 2 unique amino acid residues at positions 378 and 453 of the Fiber-2 protein might be associated with virulence due to their occurrences in all the hvFAdV-4 isolates only. To systemically evaluate the effect of age on the susceptibility of chickens to hvFAdV-4, we used this hvFAdV-4 strain to intramuscularly inoculate 7- to 180-day-old specific-pathogen-free chickens for the evaluation of pathogenicity. These results showed that the pathogenicity of the hvFAdV-4 strain GD616 to chickens exhibited age-relatedness, with younger than 59-day-old chickens showing 100% morbidity and mortality, while 180-day-old chickens still exhibited a hydropericardium syndrome-like clinicopathology with 60% morbidity and 20% mortality. These findings enrich the current available knowledge regarding the pathogenicity of the hypervirulent FAdV-4 virus in chickens with a wide range of ages, which assists with the selection of suitable-aged chickens for the evaluation of hvFAdV-4 vaccines.

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication.

Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1-38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.

Porcine Circovirus Type 3 Enters Into PK15 Cells Through Clathrin- and Dynamin-2-Mediated Endocytosis in a Rab5/Rab7 and pH-Dependent Fashion.

Porcine circovirus type 3 (PCV3) invades multiple tissues and organs of pigs of different ages and are widely spread throughout pig farms, emerging as an important viral pathogen that can potentially damage the pig industry worldwide. Since PCV3 is a newly discovered virus, many aspects of its life cycle remain unknown. Porcine kidney epithelial cells are important host targets for PCV3. Here, we used systematic approaches to dissect the molecular mechanisms underlying the cell entry and intracellular trafficking of PCV3 in PK15 cells, a cell line of porcine kidney epithelial origin. A large number of PCV3 viral particles were found to colocalize with clathrin but not caveolin-1 after entry, and PCV3 infection was significantly decreased when treated with chlorpromazine, dynasore, knockdown of clathrin heavy chain expression via RNA interference, or overexpression of a dominant-negative mutant of EPS15 in PCV3-infected cells. After internalization, the viral particles were further observed to colocalize with Rab5 and Rab7, and knockdown of both expression by RNA interference significantly inhibited PCV3 replication. We also found that PCV3 infection was impeded by ammonium chloride treatment, which indicated the requirement of an acidic environment for viral entry. Taken together, our findings demonstrate that PCV3 enters PK15 cells through a clathrin- and dynamin-2-mediated endocytic pathway, which requires early and late endosomal trafficking, as well as an acidic environment, providing an insightful theoretical basis for further understanding the PCV3 life cycle and its pathogenesis.

Fowl Adenovirus Serotype 4 Induces Hepatic Steatosis via Activation of Liver X Receptor-α.

Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic virus that causes severe hepatic damage characterized by basophilic intranuclear inclusion bodies, vacuolar degeneration, and multifocal necrosis in hepatocytes. Many aspects of FAdV-4 infection and pathogenesis, however, remain unknown. Here, we found that FAdV-4-induced hepatic injury is accompanied by the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in FAdV-4-infected chickens. Significant upregulation of adipose synthesis-related genes, such as liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding protein-1c (SREBP-1c), and significant downregulation of low-density lipoprotein secretion-related genes and lipid oxidation- and lipid decomposition-related genes were observed in the infected chickens. FAdV-4 infection in cultured leghorn male hepatoma (LMH) cells caused similar signs of steatosis, with alterations in various lipogenesis-related genes. We eliminated the effect of LXR-α activation on FAdV-4-induced steatosis and found that treatment with an LXR-α antagonist (SR9243) and RNA interference (small interfering RNA targeting LXR-α [Si-LXR-α]) decreased the number of oil droplets and the accumulation of lipogenic genes, but treatment with an LXR-α agonist (T0901317) increased the number of oil droplets and the accumulation of lipogenic genes in the cells. Additionally, SR9243 treatment or Si-LXR-α transfection led to significant reductions in viral DNA level, protein expression, and virus production, whereas T0901317 treatment caused significant increases in viral DNA level, protein expression, and virus production. However, inhibition of SREBP-1c activity had no significant effect on virus production. Collectively, these results indicated that FAdV-4-induced steatosis involves activation of the LXR-α signaling pathway, which might be a molecular mechanism underlying the hepatic injury associated with FAdV-4 infection.IMPORTANCE Fowl adenovirus serotype 4 (FAdV-4) is an important hepatotropic adenovirus in chicken, but the underlying mechanism of FAdV-4-induced hepatic injury remains unclear. We report here that infection with FAdV-4 induced the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in the livers of chickens. FAdV-4-induced steatosis might be caused by a disrupted balance of fat metabolism, as evidenced by differential regulation of various lipase genes. The significant upregulation of liver X receptor-α (LXR-α) prompted us to investigate the interplay between LXR-α activation and FAdV-4-induced steatosis. Treatment with an agonist, an antagonist, or RNA interference targeting LXR-α in cultured leghorn male hepatoma (LMH) cells indicated that FAdV-4-induced steatosis was dependent upon LXR-α activation, which contributed to virus replication. These results provide important mechanistic insights, revealing that FAdV-4 induces hepatic steatosis by activating the LXR-α signaling pathway and highlighting the therapeutic potential of strategies targeting the LXR-α pathway for the treatment of FAdV-4 infection.

Dynamic Alterations of Gut Microbiota in Porcine Circovirus Type 3-Infected Piglets.

Porcine circovirus type 3 (PCV3) is a novel porcine circovirus species associated with several diseases such as porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, cardiac pathologies, and multisystemic inflammation in piglets and sows. Currently, many studies have focused on the interaction between microbiota composition and disease progression. However, dynamic changes in the composition of the gut microbiota following PCV3 infection are still unknown. In this study, alterations in gut microbiota in PCV3-inoculated and sham-inoculated piglets were analyzed at various time points [7, 14, 21, and 28 days post-inoculation (dpi)] using the Illumina MiSeq platform. Using principal coordinate analysis, obvious structural segregations were observed in bacterial diversity and richness between PCV3- and sham-inoculated piglets, as well as at the four different time points. The abundance of gut microbiota exhibited a remarkable time-related decrease in Clostridium_sensu_stricto_1 in PCV3-inoculated piglets. In addition, significant differences were observed in functional classification based on cluster of orthologous groups assignment, between PCV3- and sham-inoculated piglets. Our findings demonstrated that PCV3 infection caused dynamic changes in the gut microbiota community. Therefore, regulating gut microbiota community may be an effective approach for preventing PCV3 infection.

Proteome Analysis in a Mammalian Cell line Reveals that PLK2 is Involved in Avian Metapneumovirus Type C (aMPV/C)-Induced Apoptosis.

Avian metapneumovirus subtype C (aMPV/C) causes an acute respiratory disease that has caused serious economic losses in the Chinese poultry industry. In the present study, we first explored the protein profile in aMPV/C-infected Vero cells using iTRAQ quantitative proteomics. A total of 921 of 7034 proteins were identified as significantly altered by aMPV/C infection. Three selected proteins were confirmed by Western blot analysis. Bioinformatics GO analysis revealed multiple signaling pathways involving cell cycle, endocytosis, and PI3K-Akt, mTOR, MAPK and p53 signaling pathways, which might participate in viral infection. In this analysis, we found that PLK2 expression was upregulated by aMPV/C infection and investigated whether it contributed to aMPV/C-mediated cellular dysfunction. Suppressing PLK2 attenuated aMPV/C-induced reactive oxygen species (ROS) production and p53-dependent apoptosis and reduced virus release. These results in a mammalian cell line suggest that high PLK2 expression correlates with aMPV/C-induced apoptosis and viral replication, providing new insight into the potential avian host cellular response to aMPV/C infection and antiviral targets.

ITRAQ-based quantitative proteomics reveals the first proteome profiles of piglets infected with porcine circovirus type 3.

Porcine circovirus type 3 (PCV3) infection induces porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammatory lesions in piglets and sows. To better understand the host responses to PCV3 infection, isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with LC-MS/MS analysis was used for quantitative determination of differentially regulated cellular proteins in the lungs of specific-pathogen-free piglets after 4 weeks of PCV3 infection. Totally, 3429 proteins were detected in three independent mass spectrometry analyses, of which 242 differential cellular proteins were significantly regulated, consisting of 100 upregulated proteins and 142 downregulated proteins in PCV3-infected group relative to control group. Bioinformatics analysis revealed that these higher or lower abundant proteins involved primarily metabolic processes, innate immune response, MHC-I and MHC-II components, and phagosome pathways. Ten genes encoding differentially regulated proteins were selected for investigation via real-time RT-PCR. The expression levels of six representative proteins, OAS1, Mx1, ISG15, IFIT3, SOD2, and HSP60, were further confirmed by Western blotting and immunohistochemistry. This study attempted for the first time to investigate the protein profile of PCV3-infected piglets using iTRAQ technology; our findings provide valuable information to better understand the mechanisms underlying the host responses to PCV3 infection in piglets. SIGNIFICANCE: Our study identified differentially abundant proteins related to a variety of potential signaling pathways in the lungs of PCV3-infected piglets. These findings provide valuable information to better understand the mechanisms of host responses to PCV3 infection.