Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells.

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

Non-cytopathic bovine viral diarrhea virus (BVDV) inhibits innate immune responses via induction of mitophagy.

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. Mitophagy plays important roles in virus-host interactions. Here, we provide evidence that non-cytopathic (NCP) BVDV shifts the balance of mitochondrial dynamics toward fission and induces mitophagy to inhibit innate immune responses. Mechanistically, NCP BVDV triggers the translocation of dynamin-related protein (Drp1) to mitochondria and stimulates its phosphorylation at Ser616, leading to mitochondrial fission. In parallel, NCP BVDV-induced complete mitophagy via Parkin-dependent pathway contributes to eliminating damaged mitochondria to inhibit MAVS- and mtDNA-cGAS-mediated innate immunity responses, mtROS-mediated inflammatory responses and apoptosis initiation. Importantly, we demonstrate that the LIR motif of ERNS is essential for mitophagy induction. In conclusion, this study is the first to show that NCP BVDV-induced mitophagy plays a central role in promoting cell survival and inhibiting innate immune responses in vitro.

Posttraumatic growth of medical staff during COVID-19 pandemic: A scoping review.

BACKGROUND: The COVID-19 pandemic has imposed unprecedented stress and challenges upon medical staff, potentially resulting in posttraumatic growth (PTG). This scoping review aims to synthesize the existing knowledge on PTG among medical staff during the pandemic by identifying its current status and potential influencing factors. The findings may provide a foundation for future research and interventions to enhance the medical staff's psychological resilience and well-being. METHODS: Literature was systematically searched on PTG among medical staff during the COVID-19 pandemic from 01 January 2020 to 31 December 2022. The following databases were searched: PubMed, Web of Science, Embase, CINAHL, PsycINFO, Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Service System (SinoMed), and Wanfang Data. Eligibility criteria included: (1) medical staff as research subjects; (2) a focus on "posttraumatic growth" or "alternative posttraumatic growth" related to the COVID-19 outbreak and pandemic; (3) discussion of the situation and influencing factors of PTG; and (4) study types, such as qualitative, quantitative, and mixed methods. Two researchers independently selected and extracted study characteristics (study design, study population, region, measurement instruments, and primary outcomes) from the included literature. The data were synthesized qualitatively and descriptively. RESULTS: Thirty-six papers from 12 countries met the inclusion criteria. Moderate PTG levels were observed among healthcare workers during the COVID-19 pandemic, with emphasis on "interpersonal relationships," "changes in life philosophy," and "growth in personal competence." Influencing factors included trauma exposure, sociodemographics, psychological characteristics (resilience and positive qualities), coping, and social support. CONCLUSIONS: This review discovered moderate PTG levels among medical staff during the COVID-19 pandemic, with critical areas in interpersonal relationships, life philosophy, and personal competence. The identified influencing factors can inform future research and interventions to enhance healthcare workers' psychological resilience and well-being.

Mitochondria-mediated ferroptosis contributes to the inflammatory responses of bovine viral diarrhea virus (BVDV) in vitro.

Bovine viral diarrhea virus (BVDV) belongs to the family Flaviviridae and includes two biotypes in cell culture: cytopathic (CP) or non-cytopathic (NCP) effects. Ferroptosis is a non-apoptotic form of programmed cell death that contributes to inflammatory diseases. However, whether BVDV induces ferroptosis and the role of ferroptosis in viral infection remain unclear. Here, we provide evidence that both CP and NCP BVDV can induce ferroptosis in Madin-Darby bovine kidney cells at similar rate. Mechanistically, biotypes of BVDV infection downregulate cytoplasmic and mitochondrial GPX4 via Nrf2-GPX4 pathway, thereby resulting in lethal lipid peroxidation and promoting ferroptosis. In parallel, BVDV can degrade ferritin heavy chain and mitochondrial ferritin via NCOA4-mediated ferritinophagy to promote the accumulation of Fe2+ and initiate ferroptosis. Importantly, CP BVDV-induced ferroptosis is tightly associated with serious damage of mitochondria and hyperactivation of inflammatory responses. In contrast, mild or unapparent damage of mitochondria and slight inflammatory responses were detected in NCP BVDV-infected cells. More importantly, different mitophagy pathways in response to mitochondria damage by both biotypes of BVDV are involved in inflammatory responses. Overall, this study is the first to show that mitochondria may play key roles in mediating ferroptosis and inflammatory responses induced by biotypes of BVDV in vitro.IMPORTANCEBovine viral diarrhea virus (BVDV) threatens a wide range of domestic and wild cattle population worldwide. BVDV causes great economic loss in cattle industry through its immunosuppression and persistent infection. Despite extensive research, the mechanism underlying the pathogenesis of BVDV remains elusive. Our data provide the first direct evidence that mitochondria-mediated ferroptosis and mitophagy are involved in inflammatory responses in both biotypes of BVDV-infected cells. Importantly, we demonstrate that the different degrees of injury of mitochondria and inflammatory responses may attribute to different mitophagy pathways induced by biotypes of BVDV. Overall, our findings uncover the interaction between BVDV infection and mitochondria-mediated ferroptosis, which shed novel light on the physiological impacts of ferroptosis on the pathogenesis of BVDV infection, and provide a promising therapeutic strategy to treat this important infectious disease with a worldwide distribution.

Fowl adenovirus serotype 4 enters leghorn male hepatocellular cells via the clathrin-mediated endocytosis pathway.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype-4 (FAdV-4) has caused large economic losses to the world poultry industry in recent years. HHS is characterized by pericardial effusion and hepatitis, manifesting as a swollen liver with focal necroses and petechial haemorrhage. However, the process of FAdV-4 entry into hepatic cells remains largely unknown. In this paper, we present a comprehensive study on the entry mechanism of FAdV-4 into leghorn male hepatocellular (LMH) cells. We first observed that FAdV-4 internalization was inhibited by chlorpromazine and clathrin heavy chain (CHC) knockdown, suggesting that FAdV-4 entry into LMH cells depended on clathrin. By using the inhibitor dynasore, we showed that dynamin was required for FAdV-4 entry. In addition, we found that FAdV-4 entry was dependent on membrane cholesterol, while neither the knockdown of caveolin nor the inhibition of a tyrosine kinase-based signalling cascade affected FAdV-4 infection. These results suggested that FAdV-4 entry required cholesterol but not caveolae. We also found that macropinocytosis played a role, and phosphatidylinositol 3-kinase (PI3K) was required for FAdV-4 internalization. However, inhibitors of endosomal acidification did not prevent FAdV-4 entry. Taken together, our findings demonstrate that FAdV-4 enters LMH cells through dynamin- and cholesterol-dependent clathrin-mediated endocytosis, accompanied by the involvement of macropinocytosis requiring PI3K. Our work potentially provides insight into the entry mechanisms of other avian adenoviruses.

ß-catenin facilitates fowl adenovirus serotype 4 replication through enhancing virus-induced autophagy.

ß-catenin is a key component of the Wnt/ß-catenin signal transduction cascade which is a highly conserved signaling pathway in eukaryotes. Increasing evidence suggests that the Wnt/ß-catenin signaling pathway is involved in the infection of many viruses. However, its role in fowl adenovirus serotype 4 (FAdV-4) replication remains unclear. In the present study, we showed that FAdV-4 infection increased the expression of ß-catenin and promoted the nuclear translocation of ß-catenin. Overexpression of ß-catenin and LiCl treatment stimulated the accumulation of ß-catenin in the nucleus, and then facilitated FAdV-4 replication. Conversely, repression of ß-catenin by inhibitors and siRNA significantly inhibited FAdV-4 replication. Furthermore, inhibition of autophagy by 3-Methyladenine (3-MA) suppressed the FAdV-4 replication, and repression of ß-catenin inhibited the FAdV-4-triggered autophagy. In conclusion, the nuclear translocation of ß-catenin benefits FAdV-4 replication, and suppression of ß-catenin limits FAdV-4 production by inhibiting FAdV-4-induced autophagy. These findings indicated that ß-catenin is an important regulator of FAdV-4 replication which can serve as a potential target for anti-FAdV-4 agents.

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen.

Mycoplasma synoviae (M. synoviae) is one of the major poultry pathogens causing infectious synovitis, airsacculitis, a high incidence of shell breakage, and egg production loss. However, the pathogenesis of M. synoviae remains unclear. Adhesion of mycoplasmas to host cells is a crucial step in infection and colonization. The purpose of this study was to determine the adhesive function of a putative P80 family lipoprotein (LP78) and evaluate its application in the detection of antibodies against M. synoviae. Recombinant LP78 (rLP78) was expressed in the supernatant component of Escherichia coli and mouse anti-rLP78 serum was prepared. Bioinformatic analysis and western blotting results revealed that LP78 was conservative among M. synoviae strains. It was distributed not only in the cytoplasm but also on the membrane of M. synoviae through western blotting and indirect immunofluorescence (IFA). The adherence of M. synoviae to DF-1 cells was significantly inhibited by mouse anti-rLP78 serum (p < 0.01). IFA revealed that rLP78 adhered to DF-1 cells, and this adherence was prevented by mouse anti-rLP78 serum. Furthermore, rLP78 was found to bind to the DF-1 cells membrane proteins in a dose-dependent manner by enzyme-linked immunosorbent assay (ELISA). Screening of DF-1 cells membrane proteins by western blotting showed that proteins with molecular weight of 35-40 kDa and 55-70 kDa bound to rLP78. Moreover, rLP78 was identified to be a fibronectin/plasminogen binding protein. The sensitivity and specificity of rLP78-based iELISA were 85.7 and 94.1%, respectively. The maximum dilution of positive serum (HI titer, 1:128) detected via rLP78-based iELISA was 1:6,400, whereas that detected using a commercial ELISA kit was 1:12,800-1:25,600. Both rLP78-based iELISA and the commercial ELISA kit detected seroconversion after 7 days of challenge and immunization. No cross-reactivity with positive sera against other avian pathogens was observed in rLP78-based iELISA. Collectively, these results indicate that LP78 is a fibronectin/plasminogen-binding adhesion protein of M. synoviae and a potential diagnostic antigen. The present study will facilitate a better understanding of the pathogenesis of M. synoviae and the development of new diagnostic.

Evaluation of the protective efficacy of six major immunogenic proteins of Mycoplasma Synoviae.

Mycoplasma synoviae (MS) is a primary avian pathogen prevalent worldwide that causes airsacculitis and synovitis in birds. Vaccination is recommended as the most cost-effective strategy in the control of MS infection. Novel alternative vaccines are needed for eradicating and controlling MS infection in flocks. DnaK, enolase, elongation factor Tu (EF-Tu), MSPB, NADH oxidase and LP78 are the major immunogenic antigens of MS and are promising targets for subunit vaccine candidates. In the present study, genes encoding DnaK, enolase, EF-Tu, MSPB, LP78, and NADH oxidase were cloned and expressed in Escherichia coli. Enzyme-linked immunosorbent assay showed that the six recombinant proteins were recognized by convalescent sera, indicating that they were expressed during infection. Two injections of the six subunit vaccines induced a robust antibody response and increased the concentrations of IFN-γ and IL-4, especially rEnolase and rEF-Tu. The proliferation of peripheral blood lymphocytes was enhanced in all of the immunized groups. Chickens immunized with rEnolase, rEF-Tu, rLP78, and rMSPB conferred significant protection against MS infection, as indicated by significantly lower DNA copies in the trachea, lower scores of air sac lesions, and lesser tracheal mucosal thickness than that in the challenge control. Especially, rEnolase provided the best protective efficacy, followed by rEF-Tu, rMSPB, and rLP78. Our finds demonstrate that the subunit vaccines and bacterin can only reduce the lesions caused by MS infection, but not prevent colonization of the organism. Our findings may contribute to the development of novel vaccine agents against MS infection.

A vesicular stomatitis virus-based African swine fever vaccine prototype effectively induced robust immune responses in mice following a single-dose immunization.

Introduction: African swine fever (ASF) is a highly contagious hemorrhagic fever disease in pigs caused by African swine fever virus (ASFV). It is very difficult to control and prevent ASF outbreaks due to the absence of safe and effective vaccines. Methods: In order to develop a safe and effective ASF vaccine for the control and prevention of ASF, two ASFV recombinant vesicular stomatitis virus (VSV) live vector vaccine prototypes, containing the gene of p72, and a chimera of p30 and p54, were developed based on the replication-competent VSV, and named VSV-p72 and VSV-p35. The immune potency of VSV-p72 or VSV-p35 alone and in combination was evaluated in BALB/c mice via intramuscular and intranasal vaccination. Results: The results indicated that whether administered alone or in combination, the two vaccine prototypes showed acceptable safety in mice and, more importantly, induced high-level specific antibodies against p72, p30, and p54 of ASFV and a strong cellular immune response 28 days after vaccination. The sera from mice vaccinated with the vaccine prototypes significantly inhibited ASFV from infecting porcine alveolar macrophages (PAMs) in vitro. Most notably, the immunized sera from a mixture of VSV-p35 and VSV-p72 inhibited ASFV from infecting PAMs, with an inhibition rate of up to 78.58%. Conclusion: Overall, our findings suggest that ASFV recombinant VSV live vector vaccine prototypes may become a promising candidate vaccine for the control and prevention of ASF.

Long noncoding RNA IRF1-AS is associated with peste des petits ruminants infection.

Peste des petits ruminants (PPR) is an acute and highly contagious disease and has long been a significant threat to small ruminant productivity worldwide. However, the molecular mechanism underlying host-PPRV interactions remains unclear and the long noncoding RNAs (lncRNAs) regulation of PPR virus (PPRV) infection has rarely been reported so far. Here, we first demonstrated that PPRV infection can induce an obvious innate immune response in caprine endometrial epithelial cells (EECs) at 48 h post-infection (hpi) with an MOI of 3. Subsequently, we determined that PPRV infection is associated with 191 significantly differentially expressed (SDE) lncRNAs, namely, 137 upregulated and 54 downregulated lncRNAs, in caprine EECs compared with mock control cells at 48 hpi by using deep sequencing technology. Importantly, bioinformatics preliminarily analyses revealed that these DE lncRNAs were closely related to the immune response. Furthermore, we identified a system of lncRNAs related to the immune response and focused on the role of lncRNA 10636385 (IRF1-AS) in regulating the innate immune response. Interestingly, we found that IRF1-AS was a potent positive regulator of IFN-ß and ISG production, which can significantly inhibit PPRV replication in host cells. In addition, our data revealed that IRF1-AS was positively correlated with its potential target gene, IRF1, which enhanced the activation of IRF3 and the expression of ISGs and interacted with IRF3. This study suggests that IRF1-AS could be a new host factor target for developing antiviral therapies against PPRV infection.

Extracellular vesicles derived from PPRV-infected cells enhance signaling lymphocyte activation molecular (SLAM) receptor expression and facilitate virus infection.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and SLAM was identified as the primary receptor for PPRV and other Morbilliviruses. Many viruses have been demonstrated to engage extracellular vesicles (EVs) to facilitate their replication and pathogenesis. Here, we provide evidence that PPRV infection significantly induced the secretion levels of EVs from goat PBMC, and that PPRV-H protein carried in EVs can enhance SLAM receptor expression in the recipient cells via suppressing miR-218, a negative miRNA directly targeting SLAM gene. Importantly, EVs-mediated increased SLAM expression enhances PPRV infectivity as well as the expression of various cytokines related to SLAM signaling pathway in the recipient cells. Moreover, our data reveal that PPRV associate EVs rapidly entry into the recipient cells mainly through macropinocytosis pathway and cooperated with caveolin- and clathrin-mediated endocytosis. Taken together, our findings identify a new strategy by PPRV to enhance virus infection and escape innate immunity by engaging EVs pathway.

Peste des petits ruminants virus induces ERS-mediated autophagy to promote virus replication.

Peste des petits ruminants virus (PPRV) has long been a significant threat to small ruminant productivity worldwide. Virus infection-induced endoplasmic reticulum (ER) stress (ERS) and the subsequently activated unfolded protein response (UPR) play significant roles in viral replication and pathogenesis. However, the relationship between ERS and PPRV infection is unknown. In this study, we demonstrated that ERS was induced during PPRV infection in caprine endometrial epithelial cells (EECs). Importantly, we demonstrated that the induction of autophagy by PPRV was mediated by ERS. Furthermore, we found that the PERK/eIF2α pathway but not the ATF6 or IRE1 pathway was activated and that the activated PERK/eIF2α pathway participated in regulating ERS-mediated autophagy. Moreover, virus replication was required for PPRV infection-induced ERS-mediated autophagy and PERK pathway activation. Additionally, we revealed that either the viral nucleocapsid (N) or nonstructural protein C was sufficient to elicit ERS and activate the PERK/eIF2α pathway, which further increased autophagy. Taken together, these results suggest that PPRV N and C protein-induced autophagy enhances viral replication through the induction of ERS and that the PERK pathway may be involved in the activation of ERS-mediated autophagy during PPRV infection.

Comparative clinical studies of primary chemoradiotherapy versus S-1 and nedaplatin chemotherapy against stage IVb oesophageal squamous cell carcinoma: a multicentre open-label randomised controlled trial.

INTRODUCTION: Oesophageal squamous cell carcinoma (OSCC) is one of the most commonly occurring devastating tumours worldwide, including in China. To date, the standard care of patients with stage IV OSCC is systemic chemotherapy and palliative care, which results in poor prognosis. However, no consensus has been established regarding the role of radiotherapy in targeting the primary tumour in patients with stage IVa OSCC. Thus, the aim of this study is to assess the effectiveness of primary radiotherapy combined with S-1 and nedaplatin (NPD) chemotherapy in the patients with stage IV OSCC. METHODS AND ANALYSIS: The study is a multicentre, open-label, randomised controlled trial. A total of 180 eligible patients with stage IV OSCC will be randomised into a study group (90 patients) and a control group (90 patients). Patients in the study group will receive radiotherapy to the primary tumour at a dose of 50.4 Gy combined with 4-6 cycles of S-1 and NPD chemotherapy. In the control group, patients will only receive 4-6 cycles of S-1 and NPD chemotherapy. The primary and secondary outcomes will be measured. The differences between the two groups will be statistically analysed with regard to overall survival, the progression-free survival and safety. All outcomes will be ascertained before treatment, after treatment and after the follow-up period.The results of this study will provide evidence on the role of radiotherapy in patients with stage IV OSCC in China, which will show new options for patients with advanced oesophageal cancer. ETHICS AND DISSEMINATION: This study was approved by the Institutional Ethics Committee of The First Hospital Affiliated of Zhengzhou University (approval number: SS-2018-04). TRIAL REGISTRATION: The trial has been registered at the Chinese Clinical Trial Registry (ChiCTR1800015765) on 1 November 2018; retrospectively registered, http://www.chictr.org.cn/index.aspx.

PPRV-Induced Autophagy Facilitates Infectious Virus Transmission by the Exosomal Pathway.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. We showed previously that PPRV induced sustained autophagy for their replication in host cells. Many studies have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate the recipient's cellular response and result in productive infection of the recipient host. Here, we show that PPRV infection results in packaging of the viral genomic RNA and partial viral proteins into exosomes of Vero cells and upregulates exosome secretion. We provide evidence showing that the exosomal viral cargo can be transferred to and establish productive infection in a new target cell. Importantly, our study reveals that PPRV-induced autophagy enhances exosome secretion and exosome-mediated virus transmission. Additionally, our data show that TSG101 may be involved in the sorting of the infectious PPRV RNA into exosomes to facilitate the release of PPRV through the exosomal pathway. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated PPRV intercellular transmission. IMPORTANCE Autophagy plays an important role in PPRV pathogenesis. The role of exosomes in viral infections is beginning to be appreciated. The present study examined the role of autophagy in secretion of infectious PPRV from Vero cells. Our data provided the first direct evidence that ATG7-mediated autophagy enhances exosome secretion and exosome-mediated PPRV transmission. TSG101 may be involved in the sorting of the infectious PPRV RNA genomes into exosomes to facilitate the release of PPRV through the exosomal pathway. Inhibition of PPRV-induced autophagy or TSG101 expression could be used as a strategy to block exosome-mediated virus transmission.

Transcriptome Analysis Reveals the Potential Role of Long Noncoding RNAs in Regulating Fowl Adenovirus Serotype 4-Induced Apoptosis in Leghorn Male Hepatocellular Cells.

Hepatitis-hydropericardium syndrome (HHS) is caused by fowl adenovirus serotype 4 (FAdV-4) and has resulted in considerable economic losses to the poultry industry globally. FAdV-4 elicits apoptosis in host cells. Long noncoding RNAs (lncRNAs) have emerged as important regulatory RNAs with profound effects on various biological processes, including apoptosis. However, it remains unknown whether lncRNAs participate in FAdV-4-induced apoptosis. In this study, RNA sequencing was applied to determine the transcription of cellular lncRNA in leghorn male hepatocellular (LMH) cells infected with FAdV-4. Cellular RNA transcription analysis demonstrated that FAdV-4 infection elicited 1798 significantly differentially expressed (DE) lncRNAs in infected LMH cells at 24 h post-infection (hpi) compared to mock control infection. In addition, 2873 DE mRNAs were also found. Target prediction and analyses revealed that 775 DE lncRNAs whose 671 target mRNAs were among the DE mRNAs were involved in several signaling pathways, including the AMPK signaling pathway, p53 signaling pathway and insulin signaling pathway. From these 775 DE lncRNAs, we identified 71 DE lncRNAs related to apoptosis based on their target gene functions. Subsequently, lncRNA 54128 was selected from the 71 identified DE lncRNAs, and its role in FAdV-4-induced apoptosis was verified. LncRNA 54128 interference significantly suppressed the rate of apoptosis, which was accompanied by reduced BMP4 transcription levels. To the best of our knowledge, this is the first study to analyze host lncRNA transcription during FAdV-4 infection. Our findings provide a better understanding of host responses to FAdV-4 infection and provide new directions for understanding the potential association between lncRNAs and FAdV-4 pathogenesis.

PPRV-induced novel miR-3 contributes to inhibit type I IFN production by targeting IRAK1.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. PPRV has evolved several mechanisms to evade IFN-I responses. We report that a novel microRNA in goat PBMCs, novel miR-3, was upregulated by PPRV to facilitate virus infection. Furthermore, PPRV V protein alone was sufficient to induce novel miR-3 expression, and NF-κB and p38 pathway may involved in the induction of novel miR-3 during PPRV infection. Importantly, we demonstrated that novel miR-3 was a potent negative regulator of IFN-α production by targeting IRAK1, which resulted in the enhancement of PPRV infection. In addition, we found that PPRV infection can activated ISGs through IFN independent and IRF3 dependent pathway. Moreover, our data revealed that novel miR-3 mediated regulation of IFN-α production may involve in the differential susceptibility between goat and sheep to PPRV. Taken together, our findings identified a new strategy taken by PPRV to escape IFN-I-mediated antiviral immune responses by engaging cellular microRNA and, thus, improve our understanding of its pathogenesis.IMPORTANCE: Peste des petits ruminants virus (PPRV) induce in the hosts a transient but severe immunosuppression, which threatens both small livestock and endangered susceptible wildlife populations in many countries. Despite extensive research has been explored, the mechanism underlying PPRV immune system evasion remains elusive. Our data provided the first direct evidence that novel microRNA-3 (novel miR-3) feedback inhibits type I IFN signaling when goat PBMCs are infected with PPRV vaccine strain N75/1, thus promoting the infection. In this study, the target of novel miR-3, IRAK1, which are important for PPRV-induced type I IFN production, have also been found. Moreover, we identified NF-κB and p38 pathways may involve in novel miR-3 induction in response to PPRV infection. Taken together, our research has provided new insight into understanding the effects of miRNA on host-virus interactions, and revealed a potential therapeutic target for antiviral intervention.

Pathogenicity and Immune Responses in Specific-Pathogen-Free Chickens During Fowl Adenovirus Serotype 4 Infection.

Hydropericardium-hepatitis syndrome, a recently emerged disease of chickens, is caused by some strains of fowl adenovirus serotype 4 (FAdV-4). However, the relationship between the immune response and cytokine expression during FAdV-4 infection is largely unknown. In this study, our data showed that all chickens exhibited typical clinical signs and lesions and that the viral load was significantly increased in both the liver and thymus following FAdV-4 infection. We also found that the appearance of tissue lesions in the liver and thymus was consistent with the viral copy numbers, indicating that virus replication in systemic organs closely correlated with disease progression. In addition, the effects of FAdV-4 infection on the transcription of some avian cytokines were studied in vivo. In general, expression of the proinflammatory cytokines interleukin (IL)-2 and interferon (IFN)-α and IFN-ß in the liver and thymus was strongly upregulated. Interestingly, the expression of IL-2 was the most highly upregulated. Expression of the anti-inflammatory cytokines IL-4, IL-10, and transforming growth factor (TGF)-ß1 and TGF-ß2, were also upregulated. Moreover, we investigated both the humoral and cellular immune responses in chickens infected with FAdV-4. Compared to those in the noninfected chickens, the antibody levels in chickens infected with FAdV-4 were significantly increased within 30 days postinfection. In addition, the ratio of CD4+/CD8+ T cells was decreased in FAdV-4-infected chickens. Taken together, these findings increase our understanding of the pathogenesis of FAdV-4 in chickens and provide a foundation for additional pathogenesis studies.

Interactions Among Expressed MicroRNAs and mRNAs in the Early Stages of Fowl Adenovirus Aerotype 4-Infected Leghorn Male Hepatocellular Cells.

Hydropericardium-hepatitis syndrome (HHS) is caused by some strains of fowl adenovirus serotype 4 (FAdV-4). However, the mechanism of FAdV-4 entry is not well understood. Therefore, to investigate the changes in host cellular response at the early stage of FAdV-4 infection, a conjoint analysis of miRNA-seq and mRNA-seq was utilized with leghorn male hepatocellular (LMH) cells at 30, 60, and 120 min after FAdV-4 infection. In total, we identified 785 differentially expressed (DE) miRNAs and 725 DE mRNAs in FAdV-4-infected LMH cells. Most miRNAs and mRNAs, including gga-miR-148a-3p, gga-miR-148a-5p, gga-miR-15c-3p, CRK, SOCS3, and EGR1, have not previously been reported to be associated with FAdV-4 infection. The conjoint analysis of the obtained data identified 856 miRNA-mRNA pairs at three time points. The interaction network analysis showed that gga-miR-128-2-5p, gga-miR-7475-5p, novel_miR205, and TCF7L1 were located in the core of the network. Furthermore, the relationship between gga-miR-128-2-5p and its target OBSL1 was confirmed using a dual-luciferase reporter system and a real-time quantitative polymerase chain reaction assay. In vitro experiments revealed that both gga-miR-128-2-5p overexpression and OBSL1 loss of function inhibited FAdV-4 entry. These results suggested that gga-miR-128-2-5p plays an important role in FAdV-4 entry by targeting OBSL1. To the best of our knowledge, the present study is the first to analyze host miRNA and mRNA expression at the early stage of FAdV-4 infection; furthermore, the results of this study help to elucidate the molecular mechanisms of FAdV-4 entry.

Autophagy induction by the pathogen receptor NECTIN4 and sustained autophagy contribute to peste des petits ruminants virus infectivity.

Macroautophagy/autophagy is an essential cellular response in the fight against intracellular pathogens. Although some viruses can escape from or utilize autophagy to ensure their own replication, the responses of autophagy pathways to viral invasion remain poorly documented. Here, we show that peste des petits ruminants virus (PPRV) infection induces successive autophagic signalling in host cells via distinct and uncoupled molecular pathways. Immediately upon invasion, PPRV induced a first transient wave of autophagy via a mechanism involving the cellular pathogen receptor NECTIN4 and an AKT-MTOR-dependent pathway. Autophagic detection showed that early PPRV infection not only increased the amounts of autophagosomes and LC3-II but also downregulated the phosphorylation of AKT-MTOR. Subsequently, we found that the binding of viral protein H to NECTIN4 ultimately induced a wave of autophagy and inactivated the AKT-MTOR pathway, which is a critical step for the control of infection. Soon after infection, new autophagic signalling was initiated that required viral replication and protein expression. Interestingly, expression of IRGM and HSPA1A was significantly upregulated following PPRV replication. Strikingly, knockdown of IRGM and HSPA1A expression using small interfering RNAs impaired the PPRV-induced second autophagic wave and viral particle production. Moreover, IRGM-interacting PPRV-C and HSPA1A-interacting PPRV-N expression was sufficient to induce autophagy through an IRGM-HSPA1A-dependent pathway. Importantly, syncytia formation could facilitate sustained autophagy and the replication of PPRV. Overall, our work reveals distinct molecular pathways underlying the induction of self-beneficial sustained autophagy by attenuated PPRV, which will contribute to improving the use of vaccines for therapy.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG: autophagy-related; BECN1: beclin 1; CDV: canine distemper virus; Co-IP: coimmunoprecipitation; FIP: fusion inhibitory peptide; GFP: green fluorescent protein; GST: glutathione S-transferase; HMOX1: heme oxygenase 1; hpi: hours post infection; HSPA1A: heat shock protein family A (Hsp70) member 1A; HSP90AA1: heat shock protein 90 kDa alpha (cytosolic), class A member 1; IFN: interferon; IgG: immunoglobulin G; INS: insulin; IRGM: immunity related GTPase M; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MeV: measles virus; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide-3 kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; SDS: sodium dodecyl sulfate; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; UV: ultraviolet.

MicroRNA-218 Regulates Signaling Lymphocyte Activation Molecular (SLAM) Mediated Peste des Petits Ruminants Virus Infectivity in Goat Peripheral Blood Mononuclear Cells.

Peste des petits ruminants virus (PPRV) has emerged as a significant threat to the productivity of small ruminants worldwide. SLAM was identified as the primary receptor for PPRV and other Morbilliviruses, although the regulation of SLAM expression is not yet fully understood. In this study, we revealed a novel mechanism by which PPRV upregulates its receptor SLAM expression and thereby benefits its replication via suppressing miR-218, a novel negative miRNA directly targeting SLAM gene. We demonstrated that PPRV infection downregulates miR-218, which in turn enhances SLAM expression on the surface of goat peripheral blood mononuclear cells (PBMCs), thus promoting PPRV replication. Since SLAM signaling may modulate the immune responses induced by PPRV infection, we further examined the effect of SLAM expression on the production of various cytokines by PBMCs in the absence or presence of PPRV. We demonstrated that miR-218-mediated SLAM expression modulates the expression of IFN-γ, TNF-α, and IL-10, importantly, these modulatory effects were enhanced in the presence of PPRV infection. Furthermore, our data clearly showed that PPRV H protein is sufficient to regulate miR-218-mediated SLAM expression. Taken together, our results suggest a novel mechanism involving post-transcriptional regulation of SLAM receptor expression on goat PBMCs during PPRV infection.