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.

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.

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.

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.

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.

The Autophagy Cargo Receptor SQSTM1 Inhibits Infectious Bursal Disease Virus Infection through Selective Autophagic Degradation of Double-Stranded Viral RNA.

Selective autophagy mediates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and protein aggregates. However, whether it targets double-stranded RNA (dsRNA) of intracellular pathogens is still largely unknown. Here, we show that selective autophagy regulates the degradation of the infectious bursal disease virus (IBDV) dsRNA genome. The amount of dsRNA decreased greatly in cells that overexpressed the autophagy-required protein VPS34 or autophagy cargo receptor SQSTM1, while it increased significantly in SQSTM1 or VPS34 knockout cells or by treating wild-type cells with the autophagy inhibitor chloroquine or wortmannin. Confocal microscopy and structured illumination microscopy showed SQSTM1 colocalized with dsRNA during IBDV infection. A pull-down assay further confirmed the direct binding of SQSTM1 to dsRNA through amino acid sites R139 and K141. Overexpression of SQSTM1 inhibited the replication of IBDV, while knockout of SQSTM1 promoted IBDV replication. Therefore, our findings reveal the role of SQSTM1 in clearing viral dsRNA through selective autophagy, highlighting the antiviral role of autophagy in the removal of the viral genome.

Very virulent infectious bursal disease virus infection caused changes in cloacal temperature and clinical manifestations in pigeons (Columba livia domestica) and is transmitted to sentinel chickens.

In this study, changes in cloacal temperature and clinical manifestations due to very virulent infectious bursal disease virus (vvIBDV) infection in pigeons (Columba livia domestica) and transmission to chickens were demonstrated. Thirty pigeons (3-6 weeks old) and thirty chickens (3 weeks old) divided into 4 groups (I-IV) were used for this study. Group I comprised of 10 uninoculated pigeons only; II comprised of 10 inoculated pigeons and 10 sentinel chickens; III comprised of 10 sentinel pigeons and 10 inoculated chickens, while IV comprised of 10 uninoculated chickens only. Pigeons in group II and chickens in group III were each inoculated with 0.20 mL (titre of 109.76CID50/mL) of vvIBDV (Nigerian strain). Cloacal temperature was monitored and clinical manifestations scored post-inoculation (pi). Results indicated significant (P < 0.05) pyrexia at 2 days pi (dpi), mild clinical signs and no mortality in inoculated pigeons. Significant (P < 0.05) pyrexia at 2-4 dpi, severe clinical signs and mortality (50%; 60%) were observed in inoculated and sentinel chickens. IBDV antigen and antibody were detected in pigeons and chickens. Pigeons showed response to vvIBDV infection thus suggesting susceptibility of pigeons to IBD. Sentinel chickens presented clinical manifestations of IBD and this suggests transmission from pigeons to chickens. This study therefore documents pyrexia and clinical manifestations due to vvIBDV infection in pigeons and successful transmission of the virus between pigeons and chickens.

Co-administration of toll-like receptor (TLR)-3 agonist Poly I:C with different infectious bursal disease (IBD) vaccines improves IBD specific immune response in chicken.

Toll-like receptor (TLR) agonists are emerging as promising vaccine adjuvants and immunomodulators in poultry against many diseases. Infectious bursa disease (IBD) still remains as a major threat to poultry industry. Improving the vaccine mediated immune response would help in better protection against IBD virus infection. Adjuvant potential of TLR3 agonist, Polinosinic polycytidylic acid (Poly I:C) with different IBD vaccines has been analyzed in chicken in the present study. Intermediate, intermediate plus IBD vaccine, bursaplex vaccine and their respective poly I:C combinations were used for immunization of chicken. IBD specific antibody titers, bursa to body weight ratio, body weight gain and bursal lesion scores were evaluated at weekly interval in different immunization groups. Fold changes in cytokines IL-1ß and IFN-γ mRNA expression levels in spleen were also analyzed in different groups. Intermediate plus IBD vaccine induced significantly (P ≤ 0.05) higher IBD specific antibody response at 35 days of age than other groups with comparatively lower body weight gain and moderate bursal lesion score. Poly I:C co-administration with intermediate IBD vaccine and bursaplex vaccine improved the IBD specific antibody titers, better body weight gain and moderately less bursal lesion score. However, Poly I:C combination with intermediate plus IBD vaccine did not improve the specific immune response. IL-1ß levels were up-regulated in intermediate plus and bursaplex group, whereas IFN-γ m RNA expression levels were upregulated in intermediate IBD with Poly I:C group. In conclusion, poly I:C co-administration with intermediate IBD and bursaplex vaccine was beneficial and improved the specific immune response with least immunosuppression and bursal damage.

Comparative growth kinetic study of Newcastle disease virus, infectious bursal disease virus and avian influenza virus in chicken embryo fibroblast and DF-1 cell lines.

Viral diseases have caused devastating effect on poultry industry leading to significant losses in economy of world. In the presented study, the ability of Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and avian influenza virus (AIV) to grow in two cell lines was evaluated. Both chicken embryo fibroblast (CEF) and DF-1 cells were used and cytopathic effects (CPE) produced by these viruses were observed. The titer of virus in terms of TCID was determined after 24h up to four days for each virus. The same type of CPE was observed for all virus- es used in the study in both DF-1 and CEF cells. IBDV showed CPE causing rounding of cells while NDV caused formation of multicellular large nuclei, cell fusion and rounding of cells. Giant cells with inclusions and aggregation of cells with intact monolayer was observed for AIV. In growth kinetic study, higher titer of IBDV and NDV was observed in CEF cells than DF-1 cells while for AIV, DF-1 cells showed higher titer than CEF cells. These results would be useful for furthers comparative studies on growth of different cell lines of various viruses to find a suitability for vaccine production.

Antox® and Bactofort® mitigated the haematological alterations induced by a very virulent infectious bursal disease virus in chicks.

The study investigated the mitigating effects of two probiotics on blood parameters of ISA Brown chicks inoculated with a very virulent infectious bursal disease virus (vvIBDV). Two hundred chicks were assigned into four groups of 50 birds each. Groups A and B were administered Antox® in water and Bactofort® in feed daily from 1 to 42 days of age and inoculated with a vvIBDV at 28 days and C and D served as positive and negative controls, respectively. Blood samples were examined for changes in packed cell volume (PCV), haemoglobin concentration (Hb), red blood cell (RBC), total white blood cell (TWBC), heterophil and lymphocyte counts seven days post inoculation. The PCV between groups A and C differed (P < 0.05) and in group B it was higher (P < 0.05) than that of group C. The Hb concentration between groups A, B and C differed (P < 0.05). There was a difference (P < 0.05) in RBC counts between groups A, B, C. Differences in TWBC between group A and C were significant (P < 0.05) and TWBC in group B was higher (P < 0.05) than that of group C. There was a significant difference in heterophil (P < 0.05) and lymphocyte (P < 0.05) count between group A and C, and B and C. Heterophil/lymphocyte ratio was significantly higher in positive control compared to groups A, B, C. Antox® and Bactofort® mitigated the deleterious effects of vvIBDV on blood parameters and can assist in cases of IBD outbreak.

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.

Dexamethasone reduces infectious bursal disease mortality in chickens.

Very virulent infectious bursal disease virus (vvIBDV) causes high mortality in chickens but measures to reduce the mortality have not been explored. Chickens (8-9 weeks) were treated with 3 agents before and during vvIBDV inoculation. Dexamethasone treatment reduced the mortality of infected chickens (40.7% vs. 3.7%; p < 0.001), but treatment with aspirin or vitamin E plus selenium did not affect the mortality. The bursa of Fabricius appeared to have shrunk in both dead and surviving chickens (p < 0.01). The results indicate that dexamethasone can reduce mortality in vvIBDV-infected chickens and may provide therapeutic clues for saving individual birds infected by the virus.

The Full Region of N-Terminal in Polymerase of IBDV Plays an Important Role in Viral Replication and Pathogenicity: Either Partial Region or Single Amino Acid V4I Substitution Does Not Completely Lead to the Virus Attenuation to Three-Yellow Chickens.

Infectious Bursal Disease Virus (IBDV) has haunted the poultry industry with severe, prolonged immunosuppression of chickens when infected at an early age and can easily lead to other secondary infections. Understanding the pathogenic mechanisms could lead to effective prevention and control of Infectious Bursal Disease (IBD). Evidence suggests that the N-terminal domain of polymerase in segment B plays an important role, but it is not clear which part or residual is crucial for the pathogenicity. Using a reverse genetics technique, a molecular clone (rNN1172) of the parental vvIBDV strain NN1172 was generated, and its pathogenicity was found to be the same as the parental virus. Then, three recombinant chimeric viruses were rescued based on the rNN1172 and substituted with the counterparts in the N-terminal domain of the attenuated vaccine strain B87: the rNN1172-B87VP1a (substituting the full region of the 1-167 aa residuals), the rNN1172-B87VP1a∆4 (substituting the region of the 5-167 aa residuals), and the rNN1172-VP1∆4 (one single aa residual substitution V4I), to better explore the role of the N-terminal domain of polymerase on the viral pathogenicity. Interestingly, all these substitutions played different roles in the viral pathogenicity: the mortality of the rNN1172-B87VP1a-challenged chickens was significantly reduced from 30% to 0%. No obvious lesion was found in the histopathological examination, and the lowest viral genome copy number was also detected in the bursa when compared to the parental and two other recombinant viruses. The mortalities caused by rNN1172-B87VP1a∆4 and rNN1172-B87VP1∆4, respectively, were all reduced to 10% and had a delayed onset of death. Our results also revealed that the pathogenicity of the IBDV was consistent with the viral replication efficiency in vivo (bursae). This study demonstrated that the full region of the N-terminal of polymerase plays an important role in viral replication and pathogenicity, but the substitutions of its partial region or a single residual do not completely lead to the virus attenuation to Three-Yellow chickens, although that significantly reduces its pathogenicity.

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.

Very virulent infectious bursal disease virus-induced immune injury is involved in inflammation, apoptosis, and inflammatory cytokines imbalance in the bursa of fabricius.

Infectious bursal disease virus (IBDV) can cause a highly contagious disease in young chickens, resulting in bursal necrosis that causes severe damage to the immune system. The effects of various IBDV strains on the bursa of Fabricius (BF) have been extensively studied; however, few studies have investigated the effects of IBDV strain LJ-5, a newly discovered very virulent IBDV (vvIBDV), infection on young chicken BF. In this study, three-week-old specific pathogen-free (SPF) chickens were infected with vvIBDV for one to five days. LJ-5 decreased the bursa index, B lymphocyte viability and immunoglobulin (Ig) levels, including IgM and IgA in the bursa and IgY in the sera. Histopathological analysis revealed necrosis and depletion of the lymphoid cells and complete loss of bursal architecture in the BF, and transmission electron microscopy revealed mitochondrial vacuoles, cristae breaks, and nuclear damage in vvIBDV-infected bursa tissue. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei significantly increased following IBDV infection. Cytokine levels increased in the bursa after IBDV infection, promoting inflammation and causing an inflammatory imbalance. Apoptotic gene expression confirmed that vvIBDV infection promotes the apoptosis of bursal cells. These results suggest that vvIBDV infection attenuate immune responses by reducing B lymphocyte activity of secretion Ig in the bursa or sera and triggers inflammation, apoptosis, and an imbalance of inflammatory cytokines in the BF, resulting in immune injury in SPF chickens, which offered basic data for further study of vvIBDV pathogenesis.

Chicken optineurin suppresses MDA5-mediated interferon ß production.

Chicken MDA5 (chMDA5), the essential accepted pattern recognition receptors for detecting cytoplasmic viral RNA in chicken, initiates interferon ß (IFN-ß) generation. However, there is an incomplete elucidation of regulating chMDA5-mediated IFN-ß production. NEMO-related protein, optineurin, was identified as inhibitors of virus triggered IFN-ß induction in human or mice. In this study, full length of chicken optineurin (chOPTN) was cloned from chicken embryo fibroblast, and its role in inhibiting IFN-ß signaling pathway was further explored. Full-length chOPTN encodes 547 amino acids residues and contains unique LC3 interaction region and ubiquitin binding domain. Chicken optineurin mRNA and protein are widely expressed in different tissues, especially the heart, kidney, and bursal fabricius (BF). Overexpressed chOPTN not only inhibits poly I:C or homos-induced human IFN-ß promoter activation in 293T cells but also suppresses poly I:C, infectious bursal disease virus (IBDV) genome double-strand RNA (dsRNA), and chMDA5-induced chicken IFN-ß (chIFN-ß) promoter activation. In addition, we first revealed that chOPTN negatively regulates chIFN-ß production via inhibiting ubiquitination of chicken TBK1, which is dependent on the ubiquitin-binding domain of chOPTN. Moreover, chIFN-ß stimulus, poly I:C, and IBDV genome dsRNA improve chOPTN expression. Endogenous chOPTN expression is also upregulated by IBDV infection in 293T, DF-1 cells, as well as in BF. Therefore, our results suggested that chOPTN plays an inhibition role of chMDA5-mediated chIFN-ß signaling pathway in chicken cells.

Critical role for G3BP1 in infectious bursal disease virus (IBDV)-induced stress granule formation and viral replication.

Stress granules (SGs), complexes for mRNA storage, are formed in host cellular response to stress stimuli and play an important role in innate immune response. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component of SGs. However, whether IBDV infection induces SG formation in host cells and what role of G3BP1 plays in this process are unclear. We report here that IBDV infection initiated typical stress granule formation and enhanced G3BP1 expression in DF-1 cells. Our data show that knockdown of G3BP1 by RNAi markedly inhibited IBDV-induced SG formation and viral replication in DF-1 cells. Conversely, ectopic expression of G3BP1 enhanced IBDV-induced SG formation and significantly promoted IBDV replication in host cells. Thus, G3BP1 plays a critical role in IBDV-induced SG formation and viral replication, providing an important clue to elucidating how IBDV employs cellular SGs for its own benefits.

Role of MicroRNAs in Host Defense against Infectious Bursal Disease Virus (IBDV) Infection: A Hidden Front Line.

Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive avian disease caused by infectious bursal disease virus (IBDV). In recent years, remarkable progress has been made in the understanding of the pathogenesis of IBDV infection and the host response, including apoptosis, autophagy and the inhibition of innate immunity. Not only a number of host proteins interacting with or targeted by viral proteins participate in these processes, but microRNAs (miRNAs) are also involved in the host response to IBDV infection. If an IBDV-host interaction at the protein level is taken imaginatively as the front line of the battle between invaders (pathogens) and defenders (host cells), their fight at the RNA level resembles the hidden front line. miRNAs are a class of non-coding single-stranded endogenous RNA molecules with a length of approximately 22 nucleotides (nt) that play important roles in regulating gene expression at the post-transcriptional level. Insights into the roles of viral proteins and miRNAs in host response will add to the understanding of the pathogenesis of IBDV infection. The interaction of viral proteins with cellular targets during IBDV infection were previously well-reviewed. This review focuses mainly on the current knowledge of the host response to IBDV infection at the RNA level, in particular, of the nine well-characterized miRNAs that affect cell apoptosis, the innate immune response and viral 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.