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.

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.