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Fowl adenovirus serotype 4 (FAdV-4) infection results in serious hepatitis-hydropericardium syndrome (HHS) in broilers, which has caused great economic losses to the poultry industry; however, the specific host responses to FAdV-4 remain unknown. In this study, we identified 141 high-confidence protein-protein interactions (PPIs) between the main viral proteins (Hexon, Fiber 1, Fiber 2, and Penton bases) and host proteins via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. We found that heat shock protein 70 (Hsp70), the protein with the highest score, and its cofactor DnaJ heat shock protein 40 family member C7 (DnaJC7) could negatively regulate the replication of FAdV-4. Furthermore, the nucleotide binding domain (NBD) of Hsp70 and the J domain of DnaJC7 were necessary for inhibiting FAdV-4 replication. We verified that DnaJC7 as a bridge could bind to Hsp70 and Hexon, assisting the indirect interaction between Hsp70 and Hexon. In addition, we found that FAdV-4 infection strongly induced the expression of autophagy proteins and cellular Hsp70 in a dose-dependent manner. Blockage of Hexon by Hsp70 overexpression was significantly reduced when the autophagy pathway was blocked by the specific inhibitor chloroquine (CQ). Our results showed that Hsp70 was co-opted by DnaJC7 to interact with viral Hexon and inhibited Hexon through the autophagy pathway, leading to a considerable restriction of FAdV-4 replication. IMPORTANCE FAdV-4, as the main cause of HHS, has quickly spread all over the world in recent years, seriously threatening the poultry industry. The aim of this study was to identify the important host proteins that have the potential to regulate the life cycle of FAdV-4. We found that Hsp70 and DnaJC7 played crucial roles in regulating the amount of viral Hexon and extracellular viral titers. Moreover, we demonstrated that Hsp70 interacted with viral Hexon with the assistance of DnaJC7, followed by suppressing Hexon protein through the autophagy pathway. These results provide new insight into the role of the molecular chaperone complex Hsp70-DnaJC7 in FAdV-4 infection and suggest a novel strategy for anti-FAdV-4 drug development by targeting the specific interactions among Hsp70, DnaJC7 and Hexon.

Since late 2011, outbreaks of pseudorabies virus (PRV) have occurred in southern China causing major economic losses to the pig industry. We previously reported that variant PRV forms and recombination in China could be the source of continued epidemics. Here, we analyzed samples from intensive pig farms in eastern China between 2017 and 2019, and sequenced the main glycoproteins (gB, gC, gD, and gE) to study the evolution characteristics of PRV. Based on the gC gene, we found that PRV variants belong to clade 2 and detected a founder effect during by the PRV epidemic. In addition, we detected inter- and intra-clade recombination; in particular, inter-clade recombination in the gB genes of strains FJ-ZXF and FJ-W2, which were recombinant with clade 1 strains. We also found specific amino-acid changes and positively selected sites, possibly associated with functional changes. This analysis of the emergence of PRV in China illustrates the need for continuous monitoring and the development of vaccines against specific variants of PRV. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00140-1) contains supplementary material, which is available to authorized users.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

Cardiac fibrosis is one of the primary mechanisms of ventricular remodeling, and there is no effective method for reversal. Activation of calcium sensing receptor (CaSR) has been reported to be involved in the development of myocardial fibrosis, but the molecular mechanism for CaSR activation has not yet been clarified and needs to be further explored. Here, we found that AngII induces cardiac fibroblast proliferation and phenotypic transformation in a dose-dependent manner with increased CaSR and autophagy related protein (Beclin1, LC3B) expression. CaSR activation results in intracellular calcium release, MEK1/2 pathway phosphorylation, autophagy activation and collagen formation induced by AngII in cardiac fibroblasts. However, pretreating the cells with Calhex231, PD98059 or 3-MA partially blocked AngII-induced cardiac fibrosis. Our data indicate that the activation of CaSR-mediated MEK/ERK and autophagic pathways is involved in AngII-induced cardiac fibrosis in vitro.

Hyperproliferation of vascular smooth muscle cells (VSMC) is a major risk factor for cardiovascular diseases. Proper mitochondrial fission and fusion is involved with VSMC function. However, the role and mechanism of mitochondrial morphological changes in VSMC proliferation are not well understood. Here, we found that calcium sensing receptor (CaSR) was increased in the aortas from spontaneous hypertensive rats (SHRs) compared with age-matched Wistar Kyoto (WKY) rats. There was also an increase in mitochondrial fission and VSMC proliferation, which was attenuated by Calhex231. In primary rat VMSC, angiotensin II (Ang II) stimulation induced cytosolic [Ca2+]i increase, mitochondrial shortening and proliferation, all of which could be attenuated by pretreatment with mitochondrial division inhibitor-1 (Mdivi-1) and Calhex231. Our data indicate that CaSR-mediated mitochondrial fission could be a therapeutic target for hyperproliferative disorders.

Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. In this study, we identified and cloned canine STING gene. Full-length STING encodes a 375 amino acid product that shares the highest similarity with feline STING. Highest levels of mRNA of canine STING were detected in the spleen and lungs while the lowest levels in the heart and muscle. Analysis of its cellular localization showed that STING is localizes to the endoplasmic reticulum. STING overexpression induced the IFN response via the IRF3 and NF-κB pathways and up-regulated the expression of ISG15 and viperin. However, knockdown of STING did not inhibit the IFN-β response triggered by poly(dA:dT), poly(I:C), or SeV. Finally, overexpression of STING significantly inhibited the replication of canine influenza virus H3N2. Collectively, our findings indicate that STING is involved in the regulation of the IFN-β pathway in canine., Highlights • Here we first identified and cloned canine STING gene. • STING overexpression induced the IFN response via the IRF3 and NF-kB pathways. • Overexpression of STING inhibited the replication of H3N2 CIV.

Calcium sensing receptor (CaSR) mediates pathological cardiac hypertrophy. Mitochondria maintain their function through fission and fusion and disruption of mitochondrial dynamic is linked to various cardiac diseases. This study examined how inhibition of CaSR by the inhibitor Calhex231 affected the mitochondrial dynamics in a hypertensive model in rats. Spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were used in this study. Cardiac function and blood pressure was evaluated at the end of the study. SHRs showed increases in the ratio of heart weight to body weight and the levels of CaSR; all of these increases were suppressed by Calhex231. Additionally, Calhex231 treatment of SHRs changed the expression of proteins involved in mitochondrial dynamics. Our results demonstrated that CaSR activation induced cardiomyocyte apoptosis through the mitochondrial dynamics mediated apoptotic pathway in hypertensive hearts.

Aims Vascular smooth muscle cell (VSMC) proliferation plays a significant role in the development of various vascular disorders. However, the effect of cortistatin (CST) on VSMC proliferation remains unclear. Therefore, the purpose of our research aimed to study whether CST protected VSMCs from angiotensin II (Ang II)-induced proliferation and which mechanisms participated in the process. Main methods Cultured rat VSMCs were treated with Ang II with or without CST for 24 h. Cell proliferation rate was measured by cell counting kit-8 (CCK8) assay. The expressions of CST and its receptors were assessed by quantitative real-time PCR (qRT-PCR). The protein expression levels were analyzed by western blots. Immunofluorescence and transmission electron microscopy (TEM) were used to observe autophagy. Key findings Our results showed that different concentrations of CST alleviated the Ang II-induced VSMC proliferation. The autophagy and reactive oxygen species (ROS) stimulated by Ang II were attenuated by CST. Furthermore, when the autophagy inhibitor 3-methyladenine (3-MA) was added, it exerted similar inhibition effects like CST, but didn't augment the protective role of CST on Ang II-induced VSMC autophagy and proliferation. Moreover, blocking somatostatin receptor 3 and 5 (SSTR3 and SSTR5) partially abrogated the suppressive effect of CST on Ang II-stimulated VSMC proliferation and autophagy. Significance This study indicated that CST could ameliorate Ang II-stimulated VSMC proliferation by inhibiting autophagy partially through its receptors SSTR3 and SSTR5, providing a reasonable evidence for CST as a novel perspective therapeutic target of vascular diseases.

Increasing evidence suggests that the NLRP3 (nucleotide oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome participates in cardiovascular diseases. However, its role and activation mechanism during hypertension remains unclear. In this study, we tested the role and mechanism of calcium-sensing receptor (CaSR) in NLRP3 inflammasome activation during hypertension. We observed that the expressions of CaSR and NLRP3 were increased in spontaneous hypertensive rats (SHRs) along with aortic fibrosis. In vascular smooth muscle cells (VSMCs), the activation of NLRP3 inflammasome associated with CaSR and collagen synthesis was induced by angiotensin II (Ang II). Furthermore, inhibition of CaSR and NLRP3 inflammasome attenuated proinflammatory cytokine release, suggesting that CaSR-mediated activation of the NLRP3 inflammasome may be a therapeutic target in aortic dysfunction and vascular inflammatory lesions.

Background Infectious bronchitis (IB) caused by the IB virus (IBV) can cause acute damage to chickens around the world. Therefore, rapid diagnosis and immune status determination are critical for controlling IBV outbreaks. Enzyme-linked immunosorbent assays (ELISAs) have been widely used in the detection of IBV antibodies in the early infection and continuous infection of IB because they are more sensitive and quicker than other diagnostic methods. Results We have developed two indirect microarray methods to detect antibodies against IBV: a chemiluminescent immunoassay test (CIT) and a rapid diagnostic test (RDT). IBV nonstructural protein 5 (nsp5) was expressed, purified from Escherichia coli, and used to spot the initiator integrated poly(dimethylsiloxane), which can provide a near “zero” background for serological assays. Compared with the IDEXX IBV Ab Test kit, CIT and RDT have a sensitivity and specificity of at least 98.88% and 91.67%, respectively. No cross-reaction was detected with antibodies against avian influenza virus subtypes (H5, H7, and H9), Newcastle disease virus, Marek’s disease virus, infectious bursal disease virus, and chicken anemia virus. The coefficients of variation of the reproducibility of the intra- and inter-assays for CIT ranged from 0.8 to 18.63%. The reproducibility of RDT was consistent with the original results. The application of the IBV nsp5 protein microarray showed that the positive rate of the CIT was 96.77%, that of the nsp5 ELISA was 91.40%, and that of the RDT was 90.32%. Furthermore, the RDT, which was visible to the naked eye, could be completed within 15 min. Our results indicated that compared with nsp5 ELISA, the CIT was more sensitive, and the RDT had similar positive rates but was faster. Furthermore, the two proposed methods were specific and stable. Conclusions Two microarray assays, which were rapid, specific, sensitive, and relatively simple, were developed for the detection of an antibody against IBV. These methods can be of great value for the surveillance of pathogens and monitoring the efficiency of vaccination.

Cyclosporin A (CsA) is an effective immunosuppressive agent, but its myocardial toxicity limits its widespread and long-term clinical application. In this study, CsA treatment led to damages in myocardial fiber structure, an increase in myocardial fibrosis, and changes in heart size and shape; moreover, the degree of damage was exacerbated with prolonged drug application and increases in dose. However, the mechanism is not clear; therefore, the purpose of this study was to reveal the mechanism of CsA-induced myocardial fibrosis and identify a new target for the prevention and treatment of CsA-induced myocardial injury. Cardiac fibroblasts were treated with CsA (5, 10, or 20 μg/mL) for 24 h. Autophagy was observed by electron microscopy and immunofluorescence. The expression of NRP-2/WDFY-1, autophagy-related proteins (Beclin1 and LC3B), fibrosis-related proteins (MMP2/9), and fibroblast phenotype conversion factor (α-SMA) was evaluated by Western blot. The expression of collagen I was determined by ELISA. Then, we used the gene interference technique to alter WDFY-1 expression with or without CsA or 3-MA treatment for 24 h, and the effects on autophagy and the expression of autophagy-related proteins, fibrosis-associated proteins, IFN-α, TNF-α, and IL-6 were determined. The results showed the following: (1) CsA induced fibrosis-related protein (MMP2/9), fibroblast phenotype conversion factor (α-SMA), and collagen I up-regulation in a dose-dependent manner. (2) CsA induced the formation of autophagosomes and up-regulated the expression of Beclin1, LC3B, and the ERK/MAPK pathway in cardiac fibroblasts. (3) CsA induced NRP-2 down-regulation and WDFY-1 up-regulation. (4) Depletion of WDFY-1 inhibited CsA-induced autophagy, TNF-α and IFN-α up-regulation, and fibrosis. (5) The autophagy inhibitor 3-MA inhibited CsA-induced TNF-α and IFN-α up-regulation and fibrosis. Overall, cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis, which promotes the progression of myocardial fibrosis.

Ethnopharmacological relevance Cyrtomium macrophyllum (Makino) Tagawa has been traditionally used as a herbal medicine for the treatment of various infectious diseases such as tapeworm infestation, colds, and viral diseases. However, no systematic study of the immunity of Cyrtomium macrophyllum ethanol extracts (CM) has yet been reported. The present work evaluates these traits. Materials and methods 120 male BALB/c mice were divided into 6 groups of 20 mice each: (1) normal group (sterile physiological saline), which served as a blank control; (2) model group (Cyclophosphamide, CY) group (sterile physiological saline), which served as a negative control; (3) low-dose CM (50 mg/kg BW); (4) intermediate-dose CM (100 mg/kg BW); (5) high-dose CM (200 mg/kg BW); (6) CM group (200 mg/kg BW). CY (0.2 ml) was administered via intraperitoneal injection. The other regimens were administered via gavage in 0.2 ml solution. Phytochemical of CM was characterized by HPLC–LTQ-Orbitrap. The acute toxicity effect of the ethanol extract of Cyrtomium macrophyllum was also investigated. Results The spleen and thymus indices of mice receiving low, intermediate, and high doses of CM recovered more quickly than those of CY mice, and they did so in a dose-dependent manner. These mice also showed higher T cell and B cell proliferation responses and macrophage function than those of CY mice, and their serum levels of interleukin-6 and interferon-γ had become normal. In acute toxicity test, CM exhibited no mortality and behavioral changes in mice. Quantitative phytochemical analysis showed flavonoids, polyphenols, and tannins to be the major compounds present in the extract, at 27.64%, 30.87%, and 11.22%, respectively. We found that 16 compounds were characterized by the interpretation of their mass spectra obtained by the MS/MS. Conclusion The current study demonstrates that Cyrtomium macrophyllum ethanol extract improved immune function in CY-treated mice.

The goal of the present study was to investigate the immune-enhancing activity of polysaccharides from Cyrtomium macrophyllum (CMP). Two experiments were carried out. In immunosuppression experiment, the immune-enhancing effect of CMP in immunosuppressive mice was performed. The results showed that CMP at high and medium doses was able to overcome the CY-induced immunosuppression, significantly increases the thymus and spleen indices, enhances lymphocyte proliferation activity and macrophage function, improves immunoglobulin and cytokines levels compared with negative control group. In macrophage immunomodulatory experiment, the immune-enhancing effect of CMP in RAW264.7 cells was measured. The results showed that CMP induced the elevation of NO production, TNF-α secretion and iNOS protein of RAW264.7 cells. CMP can also strongly increase NF-κB levels in nuclear, which is an important transcription factor that can modulate expressions of iNOS, NO and TNF-α. Therefore, the CMP could be an effective immunomodulatory agent.