The influenza virus PB2 protein evades antiviral innate immunity by inhibiting JAK1/STAT signalling.

Influenza A virus (IAV) polymerase protein PB2 has been shown to partially inhibit the host immune response by blocking the induction of interferons (IFNs). However, the IAV PB2 protein that regulates the downstream signaling pathway of IFNs is not well characterized. Here, we report that IAV PB2 protein reduces cellular sensitivity to IFNs, suppressing the activation of STAT1/STAT2 and ISGs. Furthermore, IAV PB2 protein targets mammalian JAK1 at lysine 859 and 860 for ubiquitination and degradation. Notably, the H5 subtype of highly pathogenic avian influenza virus with I283M/K526R mutations on PB2 increases the ability to degrade mammalian JAK1 and exhibits higher replicate efficiency in mammalian (but not avian) cells and mouse lung tissues, and causes greater mortality in infected mice. Altogether, these data describe a negative regulatory mechanism involving PB2-JAK1 and provide insights into an evasion strategy from host antiviral immunity employed by IAV.

Clinical Impact of Switching to Ceritinib After Severe AEs Related to Crizotinib/Alectinib in a Novel PTH2R-ALK Fusion Lung Adenocarcinoma: A Case Report.

Lung cancer is still the leading cause of morbidity and mortality by cancer among men, according to the latest epidemiological data in China. Anaplastic lymphoma kinase (ALK) rearrangements act as key oncogenic drivers of non-small cell lung cancer (NSCLC) and have been identified in 5-6% of NSCLC. Although ALK inhibitors (ALK-TKIs) were proven to be more effective than chemotherapy in ALK-positive NSCLC patients and the safety profile of these drugs was favorable, novel ALK fusions NSCLC might discontinue or switch treatment because of adverse events (AEs) have rarely previously been reported. Here, we describe a male patient with stage IV lung adenocarcinoma who carried a novel PTH2R-ALK fusion identified by next-generation sequencing (NGS). The patient first took crizotinib but switched to alectinib due to gastrointestinal AEs. Although alectinib remained effective on tumors, ceritinib (450 mg) was replaced after the AEs of hyperbilirubinemia occurred. After reducing the dose to 300mg, the diarrhea AEs caused by ceritinib were effectively relieved, and the patient obtained sustained clinical benefit with progression-free survival nearly 12 months. Our findings offer valuable information for the safety management of NSCLC patients with a novel PTH2R-ALK fusion treated by ALK-TKIs.

Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum.

The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ΔrpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.

Truncation or Deglycosylation of the Neuraminidase Stalk Enhances the Pathogenicity of the H5N1 Subtype Avian Influenza Virus in Mallard Ducks.

H5N1 subtype avian influenza virus (AIV) with a deletion of 20 amino acids at residues 49-68 in the stalk region of neuraminidase (NA) became a major epidemic virus. To determine the effect of truncation or deglycosylation of the NA stalk on virulence, we used site-directed mutagenesis to insert 20 amino acids in the short-stalk virus A/mallard/Huadong/S/2005 (SY) to recover the long-stalk virus (rSNA+). A series of short-stalk or deglycosylated-stalk viruses were also constructed basing on the long-stalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylated-stalk viruses had smaller plaques, and increased thermal and low-pH stability, and a decreased neuraminidase activity when compared with the virus rSNA+. In a mallard ducks challenge study, most of the short-stalk or deglycosylated-stalk viruses showed increased pathological lesions and virus titers in the organ tissues and increased virus shedding in the oropharynx and cloaca when compared with the rSNA+ virus, while most of the short-stalk viruses, especially rSNA-20, showed higher pathogenicity than the deglycosylated-stalk virus. In addition, the short-stalk viruses showed a significantly upregulated expression of the immune-related factors in the lungs of the infected mallard ducks, including IFN-α, Mx1, and IL-8. The results suggested that NA stalk truncation or deglycosylation increases the pathogenicity of H5N1 subtype AIV in mallard ducks, which will provide a pre-warning for prevention and control of H5N1 subtype avian influenza in the waterfowl.

Recombinant Fowlpox Virus Expressing gB Gene from Predominantly Epidemic Infectious Larygnotracheitis Virus Strain Demonstrates Better Immune Protection in SPF Chickens.

Background: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens. Antigenic mutation of infectious laryngotracheitis virus (ILTV) may result in a vaccination failure in the poultry industry and thus a protective vaccine against predominant ILTV strains is highly desirable. Methods: The full-length glycoprotein B (gB) gene of ILTV with the two mutated synonymous sites of fowlpox virus (FPV) transcription termination signal sequence was cloned into the insertion vector p12LS, which was co-transfected with wild-type (wt) FPV into chicken embryo fibroblast (CEF) to develop a recombinant fowlpox virus-gB (rFPV-gB) candidate vaccine strain. Furthermore, its biological and immunological characteristics were evaluated. Results: The results indicated that gB gene was expressed correctly in the rFPV by indirect immunofluorescent assay and Western blot, and the rFPV-gB provided a 100% protection in immunized chickens against the challenge of predominant ILTV strains that were screened by pathogenicity assay when compared with the commercialized rFPV vaccine, which only provided 83.3%. Conclusion: rFPV-gB can be used as a potential vaccine against predominant ILTV strains.

Influenza a virus antagonizes type I and type II interferon responses via SOCS1-dependent ubiquitination and degradation of JAK1.

BACKGROUND: Although influenza A virus (IAV) employs diverse strategies to evade IFN responses by inhibiting the synthesis of IFN, how IAV regulates signaling downstream of IFN is incompletely understood. METHODS: In this study, we used Western blot-based protein analysis coupled with RT-qPCR, overexpression and RNA interference to investigate the regulation of JAK1 by IAV infection. RESULTS: The results indicated that JAK1 was ubiquitinated and degraded, resulting in inhibition of type I and type II IFN responses, demonstrating that IAV antagonizes the IFN-activated JAK/STAT signaling pathway by inducing the degradation of JAK1. Furthermore. IAV infection upregulated the suppressor of cytokine signaling (SOCS) protein SOCS1, and SOCS1 mediated the ubiquitination and degradation of JAK1. CONCLUSION: Collectively, our findings suggest that IAV infection induces SOCS1 expression to promote JAK1 degradation, which in turn inhibits host innate immune responses.

Substitutions in the PB2 methionine 283 residue affect H5 subtype avian influenza virus virulence.

The influenza A virus (IAV) PB2 subunit modulates viral polymerase activity, replication kinetics and pathogenicity. Here we identified novel PB2 substitutions at position 283 of H5 subtype IAV and evaluated their biological characteristics and virulence. The substitution PB2-M283L enhanced the growth capacity and polymerase activity in human and mammalian cells in comparison to the rWT virus. The substitution PB2-M283L displayed high virulence, resulting in a greater virus load in different tissues, more severe histopathological lesions and proinflammatory cytokines burst in mice. The substitution PB2-M283I had an opposite phenotype. Our data extend the important role of PB2 substitutions in the adaptation of H5 subtype IAVs to mammalian hosts.

Luteolin Modulates SERCA2a Leading to Attenuation of Myocardial Ischemia/ Reperfusion Injury via Sumoylation at Lysine 585 in Mice.

BACKGROUND/AIMS: The myocardial sarcoplasmic reticulum calcium ATPase (SERCA2a) is a pivotal pump responsible for calcium cycling in cardiomyocytes. The present study investigated the effect of luteolin (Lut) on restoring SERCA2a protein level and stability reduced by myocardial ischemia/reperfusion (I/R) injury. We verified a hypothesis that Lut protected against myocardial I/R injury by regulating SERCA2a SUMOylation. METHODS: The hemodynamic data, myocardial infarct size of intact hearts, apoptotic analysis, mitochondrial membrane potential (ΔΨm), the level of SERCA2a SUMOylation, and the activity and expression of SERCA2a were examined in vivo and in vitro to clarify the cardioprotective effects of Lut after SUMO1 was knocked down or over-expressed. The putative SUMO conjugation sites in mouse SERCA2a were investigated as the possible regulatory mechanism of Lut. RESULTS: Initially, we found that Lut reversed the SUMOylation and stability of SERCA2a as well as the expression of SUMO1, which were reduced by I/R injury in vitro. Furthermore, Lut increased the expression and activity of SERCA2a partly through SUMO1, thus improving ΔΨm and reducing apoptotic cells in vitro and promoting the recovery of heart function and reducing infarct size in vivo. We also demonstrated that SUMO acceptor sites in mouse SERCA2a involving lysine 585, 480 and 571. Among the three acceptor sites, Lut enhanced SERCA2a stability via lysine 585. CONCLUSIONS: Our results suggest that Lut regulates SERCA2a through SUMOylation at lysine 585 to attenuate myocardial I/R injury.

Porcine reproductive and respiratory syndrome virus (PRRSV) up-regulates IL-8 expression through TAK-1/JNK/AP-1 pathways.

The acute phase of respiratory distress caused by porcine reproductive and respiratory syndrome virus (PRRSV) is likely a consequence of the release of inflammatory cytokines in the lung. IL-8, the main chemokine and activator of neutrophils, might be related to the lung injury upon PRRSV infection. In this study, we showed that PRRSV induced IL-8 expression in vivo and in vitro. Subsequently, we demonstrated that JNK and NF-κB pathways were activated upon PRRSV infection and required for the enhancement of IL-8 expression. We further verified that PRRSV-activated TAK-1 was essential for the activation of JNK and NF-κB pathways and IL-8 expression. Moreover, we revealed an AP-1 binding motif in the cloned porcine IL-8 (pIL-8) promoter, and deletion of this motif abolished the pIL-8 promoter activity. Finally, we found that the JNK-activated AP-1 subunit c-Jun was critical for the up-regulation of IL-8 expression by PRRSV. These data suggest that PRRSV-induced IL-8 production is likely through the TAK-1/JNK/AP-1 pathways.

New perspective of host microRNAs in the control of PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases in pigs, leading to significant economic losses in swine industry worldwide. PRRS virus (PRRSV) is an enveloped positive single-stranded RNA virus, which mainly infects cells of monocyte/macrophage lineage. MicroRNAs (miRNAs) are small non-coding RNAs and have emerged as important regulators of virus-host cell interactions. In the past several years, scientists have been trying to understand the interaction between host miRNAs and PRRSV infection. This review describes the mechanism of host miRNAs modulating the infection and replication of PRRSV, the approaches of delivering miRNAs into hosts, and the transcriptom of host cells during PRRSV infection. miRNAs including miR-181, miR-23, miR-30c, and miR-24-3p are reported to play important roles in PRRSV infection and replication, and in modulating host antiviral responses, while others, for example miR-204, miR-221, miR-219, need more explorations. Importantly, combining with the reverse genetic technique and current miRNAs delivery approaches such as pcDNA, recombinant lentivirus, and exosomes, we propose that miRNAs contribute to and could be used as one of potent factors in controlling PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) up-regulates IL-15 through PKCß1-TAK1-NF-κB signaling pathway.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most important infectious diseases in swine industry. IL-15 is a pleiotropic cytokine and has been shown to be essential to transform NKs, CD8 T cells, and other cells of the immune systems into functional effectors. Here, we demonstrated that the broad-spectrum or conventional PKC inhibitors repressed PRRSV-induced IL-15 expression and NF-κB activation. Subsequently, we found that the PKCß specific inhibitor inhibited PRRSV-induced IL-15 production, which was also confirmed by knock-down of PKCß1, suggesting that PKCß1 is involved in the PRRSV-induced IL-15 expression. In addition, we demonstrated that PRRSV activated NF-κB through PKCß1-induced TAK1 activation. Finally, we demonstrated that PRRSV activated PKCß1 dependent on the participation of TRIF and MAVS. These data indicate that PRRSV up-regulates IL-15 through TRIF/MAVS-PKCß1-TAK1-NF-κB signaling pathway. These findings will provide new insights into the molecular mechanisms of IL-15 production induced by PRRSV.

Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nsp4 Cleaves VISA to Impair Antiviral Responses Mediated by RIG-I-like Receptors.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant etiological agents in the swine industry worldwide. It has been reported that PRRSV infection can modulate host immune responses, and innate immune evasion is thought to play a vital role in PRRSV pathogenesis. In this study, we demonstrated that highly pathogenic PRRSV (HP-PRRSV) infection specifically down-regulated virus-induced signaling adaptor (VISA), a unique adaptor molecule that is essential for retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) signal transduction. Moreover, we verified that nsp4 inhibited IRF3 activation induced by signaling molecules, including RIG-I, MDA5, VISA, and TBK1, but not IRF3. Subsequently, we demonstrated that HP-PRRSV nsp4 down-regulated VISA and suppressed type I IFN induction. Importantly, VISA was cleaved by nsp4 and released from mitochondrial membrane, which interrupted the downstream signaling of VISA. However, catalytically inactive mutant of nsp4 abolished its ability to cleave VISA. Interestingly, nsp4 of typical PRRSV strain CH-1a had no effect on VISA. Taken together, these findings reveal a strategy evolved by HP-PRRSV to counteract anti-viral innate immune signaling, which complements the known PRRSV-mediated immune-evasion mechanisms.

Na(+)/Ca(2+) exchanger 1 (NCX-1) mediates the anti-apoptotic effect of Akt1 in neonatal rat cardiomyocytes during ischemia/reperfusion.

The purpose of this study was to investigate the anti-apoptotic role of Akt1 gene in neonatal rat cardiomyocytes and the relationship with Na(+)/Ca(2+) exchanger 1 (NCX1) during ischemia/reperfusion (IR). The cultured original rat cardiomyocytes were randomly divided into five groups: normal control group (C group), hypoxia/reoxygenation group (HR group), the control vector pLVX-EGFP-3FLAG group (CV group), the gene pLVX-EGFP-3FLAG-Akt1 transfection group (A group), and Akt1 inhibitor LY294002 group (LY group). Cardiomyocyte vitality was determined using MTT, and the apoptosis was determined by TUNEL to verify the anti-apoptotic role of Akt1. The mRNA levels of Akt1 and NCX1 were determined by RT-PCR, the protein expression of Akt1, p-Akt1, NCX1 and the apoptotic proteins of mitochondrial pathway cytochrome C (Cyto C) and caspase-9 were measured by Western blot. As a result, transfected Akt1 (A group) showed increased myocardial cell viability and reduced apoptosis, with increase in Akt1 expression and decrease in NCX1 expression. The levels of apoptotic proteins Cyto C and caspase-9 also declined. This study demonstrated that lentivirus-mediated transfection of Akt1 played an anti-apoptotic role during IR of rat cardiomyocytes, via inhibition of NCX1 and other mitochondrial proteins.

Cordycepin enhances Epstein-Barr virus lytic infection and Epstein-Barr virus-positive tumor treatment efficacy by doxorubicin.

The consistent latent presence of Epstein-Barr virus (EBV) in tumor cells offers potential for virus-targeted therapies. The switch from the latent form of EBV to the lytic form in tumor cells can lead to tumor cell lysis. In this study, we report that a natural small molecule compound, cordycepin, can induce lytic EBV infection in tumor cells. Subsequently, we demonstrate that cordycepin can enhance EBV reactivating capacity and EBV-positive tumor cell killing ability of low dose doxorubicin. The combination of cordycepin and doxorubicin phosphorylates CCAAT/enhancer binding protein ß (C/EBPß) through protein kinase C (PKC)-p38 mitogen activated protein kinases (p38 MAPK) signaling pathway, and C/EBPß is required for the activation of lytic EBV infection. Most importantly, an in vivo experiment demonstrates that the combination of cordycepin and doxorubicin is more effective in inhibiting tumor growth in SCID mice than is doxorubicin alone. Our findings establish that cordycepin can enhance the efficacy of conventional chemotherapy for treatment of EBV-positive tumors.

Salvianolic Acid A Attenuates Cell Apoptosis, Oxidative Stress, Akt and NF-κB Activation in Angiotensin-II Induced Murine Peritoneal Macrophages.

We discuss the role of Salvianolic acid A(SAA), one of the main effective components in Salvia Miltiorrhiza (known as 'Danshen' in traditional Chinese medicine), in apoptotic factors, the production of oxidative products, and the expression of Akt and NF-κB in angiotensin II (Ang II)-mediated murine macrophages. In the present study, Ang II was added to mice abdominal macrophages with or without addition of SAA. After cell identification, apoptosis was measured by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining, and the expression of Bcl-2 and Bax. Intracellular concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA) were also measured. Western blotting determined the expression of Akt, p-Akt, NF-κB and p-NF-κB. Ly294002 (the inhibitor of PI3K) was used to determine the mechanism of SAA. Ang II (1 µM) significantly increased the number of TUNEL-positive cells and Bax expression, but reduced Bcl-2 expression. These effects were antagonized when the cells were pretreated with SAA. SAA decreased MDA, but increased SOD in the cell lysis solution treated with Ang II. It markedly reduced the level of p-NF-κB, as also p-Akt, which was partly blocked by Ly294002. SAA prevents Ang IIinduced apoptosis, oxidative stress and related protein expression in the macrophages. It also inhibits the activation of Akt.