OASL suppresses infectious bursal disease virus replication by targeting VP2 for degrading through the autophagy pathway.

Infectious bursal disease (IBD) is an acute and fatal immunosuppressive disease caused by infectious bursal disease virus (IBDV). As an obligate intracellular parasite, IBDV infection is strictly regulated by host factors. Knowledge on the antiviral activity and possible mechanism of host factors might provide the theoretical basis for the prevention and control of IBD. In this study, RNA-sequencing results indicated that many host factors were induced by IBDV infection, among which the expression levels of OASL (2´,5´-oligadenylate synthetase-like protein) was significantly upregulated. OASL overexpression significantly inhibited IBDV replication, whereas OASL knockdown promoted IBDV replication. Interestingly, the antiviral ability of OASL was independent of its canonical enzymatic activity, i.e., OASL targeted viral protein VP2 for degradation, depending on the autophagy receptor p62/SQSTM1 in the autophagy pathway. Additionally, the 316 lysine (K) of VP2 was the key site for autophagy degradation, and its replacement with arginine disrupted VP2 degradation induced by OASL and enhanced IBDV replication. Importantly, our results for the first time indicate a unique and potent defense mechanism of OASL against double-stranded RNA virus by interaction with viral proteins, which leads to their degradation. IMPORTANCE: OASL (2´,5´-oligadenylate synthetase-like protein) exhibits broad-spectrum antiviral effects against single-stranded RNA viruses in mammals, potentially serving as a promising target for novel antiviral strategies. However, its role in inhibiting the replication of double-stranded RNA viruses (dsRNA viruses), such as infectious bursal disease virus (IBDV), in avian species remains unclear. Our findings indicated a unique and potent defense mechanism of OASL against dsRNA viruses. It has been previously shown in mammals that OASL inhibits virus replication through increasing interferon production. The groundbreaking aspect of our study is the finding that OASL has the ability to interact with IBDV viral protein VP2 and target it for degradation and thus exerts its antiviral effect. Our results reveal the interaction between avian natural antiviral immune response and IBDV infection. Our study not only enhances our understanding of bird defenses against viral infections but can also inform strategies for poultry disease management.

Reduced NR2F2 Expression in the Host Response to Infectious Bursal Disease Virus Infection Suppressed Viral Replication by Enhancing Type I Interferon Expression by Targeting SOCS5.

Nuclear receptors are ligand-activated transcription factors that play an important role in regulating innate antiviral immunity and other biological processes. However, the role of nuclear receptors in the host response to infectious bursal disease virus (IBDV) infection remains elusive. In this study, we show that IBDV infection or poly(I·C) treatment of DF-1 or HD11 cells markedly decreased nuclear receptor subfamily 2 group F member 2 (NR2F2) expression. Surprisingly, knockdown, knockout, or inhibition of NR2F2 expression in host cells remarkably inhibited IBDV replication and promoted IBDV/poly(I·C)-induced type I interferon and interferon-stimulated genes expression. Furthermore, our data show that NR2F2 negatively regulates the antiviral innate immune response by promoting the suppressor of cytokine signaling 5 (SOCS5) expression. Thus, reduced NR2F2 expression in the host response to IBDV infection inhibited viral replication by enhancing the expression of type I interferon by targeting SOCS5. These findings reveal that NR2F2 plays a crucial role in antiviral innate immunity, furthering our understanding of the mechanism underlying the host response to viral infection. IMPORTANCE Infectious bursal disease (IBD) is an immunosuppressive disease causing considerable economic losses to the poultry industry worldwide. Nuclear receptors play an important role in regulating innate antiviral immunity. However, the role of nuclear receptors in the host response to IBD virus (IBDV) infection remains elusive. Here, we report that NR2F2 expression decreased in IBDV-infected cells, which consequently reduced SOCS5 expression, promoted type I interferon expression, and suppressed IBDV infection. Thus, NR2F2 serves as a negative factor in the host response to IBDV infection by regulating SOCS5 expression, and intervention in the NR2F2-mediated host response by specific inhibitors might be employed as a strategy for prevention and treatment of IBD.

IFITM3 reduces infectious bursal disease virus proliferation by regulating interferon expression.

Interferon-inducible transmembrane protein 3 (IFITM3), a member of the interferon-stimulating factor (ISG) family, has various antiviral functions. Infectious bursal disease virus (IBDV) mainly invades the bursa of Fabricius in chickens, causing a reduction in their immunity and resulting in death from secondary infections. Our previous study found that IBDV infection promotes the expression of chicken IFITM3. However, the role of chicken IFITM3 in IBDV infection remains unknown. To explore this role, the overexpression vector for IFITM3 was constructed and transfected into HD-11 and DF-1 cells. The results showed that the overexpression of IFITM3 significantly reduced IBDV proliferation. While the IBDV proliferation increased when IFITM3 was inhibited by using siRNA. To further explore the mechanism by which IFITM3 reduces IBDV proliferation, the effects of IFITM3 on interferon (IFN) were investigated. Transfecting the constructed IFITM3 vectors into HD-11 and DF-1 cells demonstrated that IFITM3 promoted the expression of IFN-α, IFN-ß, and IFN-γ. To investigate the mechanism by which IFITM3 regulates IFN expression, the effects of IFITM3 on IFN production were explored. The results showed that the IKB gene mainly affected the regulatory effects of IFITM3 on IFN. Taken together, IFITM3 may reduce viral proliferation by regulating changes in IFNs, and this process may involve a positive feedback effect of IFITM3 on IFN. IKB plays an important role in the regulation of IFN effects by IFITM3.

Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

Identification of Chicken CD44 as a Novel B Lymphocyte Receptor for Infectious Bursal Disease Virus.

Infectious bursal disease virus (IBDV), which targets bursa B lymphocytes, causes severe immunosuppressive disease in chickens, inducing huge economic losses for the poultry industry. To date, the functional receptor for IBDV binding and entry into host cells remains unclear. This study used mass spectrometry to screen host proteins of chicken bursal lymphocytes interacting with VP2. The chicken transmembrane protein cluster of differentiation 44 (chCD44) was identified and evaluated for its interaction with IBDV VP2, the major capsid protein. Overexpression and knockdown experiments showed that chCD44 promotes replication of IBDV. Furthermore, soluble chCD44 and the anti-chCD44 antibody blocked virus binding. The results of receptor reconstitution indicated that chCD44 overexpression conferred viral binding capability in nonpermissive cells. More important, although we found that IBDV could not replicate in the chCD44-overexpressed nonpermissive cells, the virus could enter nonpermissive cells using chCD44. Our finding reveals that chCD44 is a cellular receptor for IBDV, facilitating virus binding and entry in target cells by interacting with the IBDV VP2 protein. IMPORTANCE Infectious bursal disease virus (IBDV) causes severe immunosuppressive disease in chickens, inducing huge economic losses for the poultry industry. However, the specific mechanism of IBDV invading host cells of IBDV was not very clear. This study shed light on which cellular protein component IBDV is used to bind and/or enter B lymphocytes. The results of our study revealed that chCD44 could promote both the binding and entry ability of IBDV in B lymphocytes, acting as a cellular receptor for IBDV. Besides, this is the first report about chicken CD44 function in viral replication. Our study impacts the understanding of the IBDV binding and entry process and sets the stage for further elucidation of the infection mechanism of IBDV.

GATA3 Inhibits Viral Infection by Promoting MicroRNA-155 Expression.

Recognition of viral RNAs by melanoma differentiation associated gene-5 (MDA5) initiates chicken antiviral response by producing type I interferons. Our previous studies showed that chicken microRNA-155-5p (gga-miR-155-5p) enhanced IFN-ß expression and suppressed the replication of infectious burse disease virus (IBDV), a double-stranded RNA (dsRNA) virus causing infectious burse disease in chickens. However, the mechanism underlying IBDV-induced gga-miR-155-5p expression in host cells remains elusive. Here, we show that IBDV infection or poly(I:C) treatment of DF-1 cells markedly increased the expression of GATA-binding protein 3 (GATA3), a master regulator for TH2 cell differentiation, and that GATA3 promoted gga-miR-155-5p expression in IBDV-infected or poly(I:C)-treated cells by directly binding to its promoter. Surprisingly, ectopic expression of GATA3 significantly reduced IBDV replication in DF-1 cells, and this reduction could be completely abolished by treatment with gga-miR-155-5p inhibitors, whereas knockdown of GATA3 by RNA interference enhanced IBDV growth, and this enhancement could be blocked with gga-miR-155-5p mimics, indicating that GATA3 suppressed IBDV replication by gga-miR-155-5p. Furthermore, our data show that MDA5 is required for GATA3 expression in host cells with poly(I:C) treatment, so are the adaptor protein TBK1 and transcription factor IRF7, suggesting that induction of GATA3 expression in IBDV-infected cells relies on MDA5-TBK1-IRF7 signaling pathway. These results uncover a novel role for GATA3 as an antivirus transcription factor in innate immune response by promoting miR-155 expression, further our understandings of host response against pathogenic infection, and provide valuable clues to the development of antiviral reagents for public health. IMPORTANCE Gga-miR-155-5p acts as an important antivirus factor against IBDV infection, which causes a severe immunosuppressive disease in chicken. Elucidation of the mechanism regulating gga-miR-155-5p expression in IBDV-infected cells is essential to our understandings of the host response against pathogenic infection. This study shows that transcription factor GATA3 initiated gga-miR-155-5p expression in IBDV-infected cells by directly binding to its promoter, suppressing viral replication. Furthermore, induction of GATA3 expression was attributable to the recognition of dsRNA by MDA5, which initiates signal transduction via TBK1 and IRF7. Thus, it is clear that IBDV induces GATA3 expression via MDA5-TBK1-IRF7 signaling pathway, thereby suppressing IBDV replication by GATA3-mediated gga-miR-155-5p expression. This information remarkably expands our knowledge of the roles for GATA3 as an antivirus transcription factor in host innate immune response particularly at an RNA level and may prove valuable in the development of antiviral drugs for public health.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Phosphatidylinositol 3-Phosphate Mediates the Establishment of Infectious Bursal Disease Virus Replication Complexes in Association with Early Endosomes.

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCEInfectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.

MDA5 attenuate autophagy in chicken embryo fibroblasts infected with IBDV.

1. The role of melanoma differentiation-associated protein 5 (MDA5) in infectious bursal disease virus (IBDV)-induced autophagy was studied in chicken embryos.2. Chicken embryo fibroblasts (CEF) were used as the research model and small interfering RNA (siRNA), western blot, indirect enzyme-linked immunosorbent assay (ELISA), real-time fluorescence quantitative polymerase chain reaction (PCR) and transmission electron microscopy were used to detect autophagy, IBDV replication, CEF damage, and activation of both MDA5 and its signalling pathway.3. The results showed that CEF infected with IBDV activated the intracellular MDA5 signalling pathway and caused autophagy via inactivation of the AKT/mTOR pathway. While autophagy promotes IBDV proliferation, MDA5 weakens IBDV-induced CEF autophagy thus inhibiting IBDV replication and protecting CEF cells.4. The results indicated that chMDA5 can be activated by IBDV and attenuate CEF autophagy caused by IBDV infection, thereby inhibiting IBDV replication. This study provided a foundation for further exploring the relationship between viruses, autophagy and the pathogenic mechanism of the MDA5 pathway involved in IBDV.

Epigenetic Upregulation of Chicken MicroRNA-16-5p Expression in DF-1 Cells following Infection with Infectious Bursal Disease Virus (IBDV) Enhances IBDV-Induced Apoptosis and Viral Replication.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets and play important roles in the host response to pathogenic infection. Although infectious bursal disease virus (IBDV)-induced apoptosis in host cells has been established, the underlying molecular mechanism is not completely unraveled. Here, we show that infection of DF-1 cells by IBDV induced gga-miR-16-5p (chicken miR-16-5p) expression via demethylation of the pre-miR-16-2 (gga-miR-16-5p precursor) promoter. We found that ectopic expression of gga-miR-16-5p in DF-1 cells enhanced IBDV-induced apoptosis by directly targeting the cellular antiapoptotic protein B-cell lymphoma 2 (Bcl-2), facilitating IBDV replication in DF-1 cells. In contrast, inhibition of endogenous miR-16-5p markedly suppressed apoptosis associated with enhanced Bcl-2 expression, arresting viral replication in DF-1 cells. Furthermore, infection of DF-1 cells with IBDV reduced Bcl-2 expression, and this reduction could be abolished by inhibition of gga-miR-16-5p expression. Moreover, transfection of DF-1 cells with gga-miR-16-5p mimics enhanced IBDV-induced apoptosis associated with increased cytochrome c release and caspase-9 and -3 activation, and inhibition of caspase-3 decreased IBDV growth in DF-1 cells. Thus, epigenetic upregulation of gga-miR-16-5p expression by IBDV infection enhances IBDV-induced apoptosis by targeting the cellular antiapoptotic protein Bcl-2, facilitating IBDV replication in host cells.IMPORTANCE Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive disease in young chickens, causing severe economic losses to stakeholders across the globe. Although IBD virus (IBDV)-induced apoptosis in the host has been established, the underlying mechanism is not very clear. Here, we show that infection of DF-1 cells by IBDV upregulated gga-miR-16-5p expression via demethylation of the pre-miR-16-2 promoter. Overexpression of gga-miR-16-5p enhanced IBDV-induced apoptosis associated with increased cytochrome c release and caspase-9 and -3 activation. Importantly, we found that IBDV infection induced expression of gga-miR-16-5p that triggered apoptosis by targeting Bcl-2, favoring IBDV replication, while inhibition of gga-miR-16-5p in IBDV-infected cells restored Bcl-2 expression, slowing down viral growth, indicating that IBDV induces apoptosis by epigenetic upregulation of gga-miR-16-5p expression. These findings uncover a novel mechanism employed by IBDV for its own benefit, which may be used as a potential target for intervening IBDV infection.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens.

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1ß, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.

Ubiquitination Is Essential for Avibirnavirus Replication by Supporting VP1 Polymerase Activity.

Ubiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We report that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV). In vivo detection revealed the ubiquitination of VP1 protein in IBDV-infected target organs and different cells but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C terminus. The K751 ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C terminus of VP1 enhanced its polymerase activity. The K751-to-R mutation of VP1 protein did not block the rescue of IBDV but decreased the replication ability of IBDV. Our data demonstrate that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.IMPORTANCE Avibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression, and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C terminus of VP1 is the target site for ubiquitin, and its ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantly impairs virus replication. We conclude that VP1 is the ubiquitin-modified protein and reveal the mechanism by which VP1 promotes avibirnavirus replication.

SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1.

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues 404I and 406I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues 404I and 406I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.

Effects of infectious bursal disease virus infection on interferon and antiviral gene expression in layer chicken bursa.

Layer chickens were artificially challenged with infectious bursal disease virus (IBDV), and the kinetics of IFN-λ and antiviral genes in the bursa were explored using quantitative real-time PCR. Data showed that after the chickens were infected with IBDV, the virus load in the bursa of the Fabricius peaked at 96 h and gradually decreased. The relative mRNA expression levels of IFN-λ and antiviral genes (zinc-finger antiviral protein [ZAP], interferon alpha-inducible protein 6 [IFI6], laboratory of genetics and physiology 2 [LGP2], virus inhibitory protein [Viperin], and Mx) of the infected group dramatically increased at 24-168 h compared with those of the negative-infected group. Furthermore, the ZAP mRNA expression peaked at 24 h (3.97-fold). The Viperin mRNA transcript level was highest at 48 h (384.60-fold). The mRNA expression levels of IFI6 (96.31-fold), LGP2 (18.29-fold), and Mx (88.85-fold) peaked at 72 h, and that of IFN-λ was most remarkable at 96 h (2978.81-fold). Furthermore, the ZAP change rule was significantly positively correlated with the change rule of the IBDV load. The mRNA expression levels of IFN-λ and antiviral genes (ZAP, IFI6, LGP2, Viperin, and Mx) increased as the virus expression increased and then decreased. These results further corroborated that the IBDV infection seriously interfered with the chicken's innate immune response.

VP1 and VP3 Are Required and Sufficient for Translation Initiation of Uncapped Infectious Bursal Disease Virus Genomic Double-Stranded RNA.

Infectious bursal disease virus (IBDV) is a bisegmented double-strand RNA (dsRNA) virus of the Birnaviridae family. While IBDV genomic dsRNA lacks a 5' cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts were directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by trans-supplementation of the viral proteins VP1 and VP3 which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5' and 3' untranslated regions (UTRs) of IBDV dsRNA were essential for VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, downregulation or inhibition of the cap-binding protein eIF4E did not decrease but, rather, enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of the 5' cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.IMPORTANCE A key point of control for virus replication is viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery and is thus noninfectious. Our results constitute the first direct experimental evidence that VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute for cap and replacing the cap-binding protein eIF4E. Significantly, VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense strand but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strands of the viral genomic dsRNA.

MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally through silencing or degrading their targets, thus playing important roles in the immune response. However, the role of miRNAs in the host response against infectious bursal disease virus (IBDV) infection is not clear. In this study, we show that the expression of a series of miRNAs was significantly altered in DF-1 cells after IBDV infection. We found that the miRNA gga-miR-130b inhibited IBDV replication via targeting the specific sequence of IBDV segment A and enhanced the expression of beta interferon (IFN-ß) by targeting suppressors of cytokine signaling 5 (SOCS5) in host cells. These findings indicate that gga-miR-130b-3p plays a crucial role in host defense against IBDV infection.IMPORTANCE This work shows that gga-miR-130b suppresses IBDV replication via directly targeting the viral genome and cellular SOCS5, the negative regulator for type I interferon expression, revealing the mechanism underlying gga-miR-130-induced inhibition of IBDV replication. This information will be helpful for the understanding of how host cells combat pathogenic infection by self-encoded small RNA and furthers our knowledge of the role of microRNAs in the cell response to viral infection.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.

Infectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-Å-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) viruses.IMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages.

Infectious bursal disease virus tissue tropism and pathogenesis of the infection in chickens by application of in situ PCR, immunoperoxase and HE staining.

Infectious bursal disease is one of an OIE list of notifiable diseases. Chicken is the only host that manifests clinical signs and its pathogenicity is correlated with the distribution of antigens in organs. This study was conducted to determine disease pathogenesis and virus tissue tropism by in situ PCR, immunoperoxidase staining (IPS), and HE staining. Twenty four chickens were infected with very virulent Infectious Bursal Disease Virus (vvIBDV). Fifteen chickens were kept as a control group. Infected chickens were sacrificed at hrs 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). While, control chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Different tissues were collected, fixed in 10% buffered formalin, and processed. At hr 2 pi, virus was detected in intestinal, junction of the proventriculus and gizzard, cecal tonsil, liver, kidney, and bursa of Fabricius. At hr 4 pi, virus reached spleen, and at hr 6 pi, it entered thymus. At hr 12 pi, virus concentration increased in positive tissues. The latest invaded tissue was muscle on day 1 pi. Secondary viraemia occurred during 12-24 h pi. In situ PCR was the most sensitive technique to highlight obscure points of infection in this study.

The Roles of MicroRNAs (miRNAs) in Avian Response to Viral Infection and Pathogenesis of Avian Immunosuppressive Diseases.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of various biological processes including cell development and differentiation, apoptosis, tumorigenesis, immunoregulation and viral infections. Avian immunosuppressive diseases refer to those avian diseases caused by pathogens that target and damage the immune organs or cells of the host, increasing susceptibility to other microbial infections and the risk of failure in subsequent vaccination against other diseases. As such, once a disease with an immunosuppressive feature occurs in flocks, it would be difficult for the stakeholders to have an optimal economic income. Infectious bursal disease (IBD), avian leukemia (AL), Marek's disease (MD), chicken infectious anemia (CIA), reticuloendotheliosis (RE) and avian reovirus infection are on the top list of commonly-seen avian diseases with a feature of immunosuppression, posing an unmeasurable threat to the poultry industry across the globe. Understanding the pathogenesis of avian immunosuppressive disease is the basis for disease prevention and control. miRNAs have been shown to be involved in host response to pathogenic infections in chickens, including regulation of immunity, tumorigenesis, cell proliferation and viral replication. Here we summarize current knowledge on the roles of miRNAs in avian response to viral infection and pathogenesis of avian immunosuppressive diseases, in particular, MD, AL, IBD and RE.