Infectious Bursal Disease Virus Replication Research Articles

The down-regulation of casein kinase 1 alpha as a host defense response against infectious bursal disease virus infection

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. Although the gene functions of IBDV have been well characterized, the host responses during IBDV infection remain much poor. In the present study, casein kinase 1 alpha (CK1α), a novel VP2-associated protein, was down-regulated during IBDV replication in DF1 cells. Further experiments showed that siRNA-mediated knockdown of CK1α inhibited IBDV replication, while overexpression of CK1α promoted IBDV growth. Finally, we revealed that the effects of CK1α expression level on IBDV replication were involved in the negative regulation of CK1α on type I interferon receptor (IFNAR1), because ubiquitination assay analyses demonstrated that CK1α could promote the ubiquitination of IFNAR1, thereby affecting the stability of this receptor. In conclusion, down-regulation of CK1α during IBDV infection as a host defense response increased abundance of IFNAR1, which in turn enhanced an inhibitory effect on IBDV replication.

Infectious Bursal Disease Virus Subverts Autophagic Vacuoles To Promote Viral Maturation and Release.

Autophagy functions as an intrinsic antiviral defense. However, some viruses can subvert or even enhance host autophagic machinery to increase viral replication and pathogenesis. The role of autophagy during avibirnavirus infection, especially late stage infection, remains unclear. In this study, infectious bursal disease virus (IBDV) was used to investigate the role of autophagy in avibirnavirus replication. We demonstrated IBDV induction of autophagy as a significant increase in puncta of LC3+ autophagosomes, endogenous levels of LC3-II, and ultrastructural characteristics typical of autophagosomes during the late stage of infection. Induction of autophagy enhances IBDV replication, whereas inhibition of autophagy impairs viral replication. We also demonstrated that IBDV infection induced autophagosome-lysosome fusion, but without active degradation of their contents. Moreover, inhibition of fusion or of lysosomal hydrolysis activity significantly reduced viral replication, indicating that virions utilized the low-pH environment of acidic organelles to facilitate viral maturation. Using immuno-transmission electron microscopy (TEM), we observed that a large number of intact IBDV virions were arranged in a lattice surrounded by p62 proteins, some of which lay between virions. Additionally, many virions were encapsulated within the vesicular membranes, with an obvious release stage observed by TEM. The autophagic endosomal pathway facilitates low-pH-mediated maturation of viral proteins and membrane-mediated release of progeny virions.IMPORTANCE IBDV is the most extensively studied virus in terms of molecular characteristics and pathogenesis; however, mechanisms underlying the IBDV life cycle require further exploration. The present study demonstrated that autophagy enhances viral replication at the late stage of infection, and the autophagy pathway facilitates IBDV replication complex function and virus assembly, which is critical to completion of the virus life cycle. Moreover, the virus hijacks the autophagic vacuoles to mature in an acidic environment and release progeny virions in a membrane-mediated cell-to-cell manner. This autophagic endosomal pathway is proposed as a new mechanism that facilitates IBDV maturation, release, and reinternalization. This report presents a concordance in exit strategies among some RNA and DNA viruses, which exploit autophagy pathway for their release from cells.

Immunomodulatory and Anti-IBDV Activities of the Polysaccharide AEX from Coccomyxa gloeobotrydiformis.

A number of polysaccharides have been reported to show immunomodulatory and antiviral activities against various animal viruses. AEX is a polysaccharide extracted from the green algae, Coccomyxa gloeobotrydiformis. The aim of this study was to examine the function of AEX in regulating the immune response in chickens and its capacity to inhibit the infectious bursal disease virus (IBDV), to gain an understanding of its immunomodulatory and antiviral ability. Here, preliminary immunological tests in vitro showed that the polysaccharide AEX can activate the chicken peripheral blood molecular cells’ (PBMCs) response by inducing the production of cytokines and NO, promote extracellular antigen presentation but negatively regulate intracellular antigen presentation in chicken splenic lymphocytes, and promote the proliferation of splenic lymphocytes and DT40 cells. An antiviral analysis showed that AEX repressed IBDV replication by the deactivation of viral particles or by interfering with adsorption in vitro and reduced the IBDV viral titer in the chicken bursa of Fabricius. Finally, in this study, when AEX was used as an adjuvant for the IBDV vaccine, specific anti-IBDV antibody (IgY, IgM, and IgA) titers were significantly decreased. These results indicate that the polysaccharide AEX may be a potential alternative approach for anti-IBDV therapy and an immunomodulator for the poultry industry. However, more experimentation is needed to find suitable conditions for it to be used as an adjuvant for the IBDV vaccine.

A free VP3 C-terminus is essential for the replication of infectious bursal disease virus

Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3’ noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3’ NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication.

Non-Lytic Egression of Infectious Bursal Disease Virus (IBDV) Particles from Infected Cells.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a devastating immunosuppressive disease affecting juvenile domestic chickens. IBDV particles are naked icosahedrons enclosing a bipartite double-stranded RNA genome harboring three open reading frames (ORF). One of these ORFs codes for VP5, a non-structural polypeptide dispensable for virus replication in tissue culture but essential for IBDV pathogenesis. Using two previously described recombinant viruses, whose genomes differ in a single nucleotide, expressing or not the VP5 polypeptide, we have analyzed the role of this polypeptide during the IBDV replication process. Here, we show that VP5 is not involved in house-keeping steps of the virus replication cycle; i.e. genome transcription/replication, protein translation and virus assembly. Although infection with the VP5 expressing and non-expressing viruses rendered similar intracellular infective progeny yields, striking differences were detected on the ability of their progenies to exiting infected cells. Experimental data shows that the bulk of the VP5-expressing virus progeny efficiently egresses infected cells during the early phase of the infection, when viral metabolism is peaking and virus-induced cell death rates are as yet minimal, as determined by qPCR, radioactive protein labeling and quantitative real-time cell death analyses. In contrast, the release of the VP5-deficient virus progeny is significantly abridged and associated to cell death. Taken together, data presented in this report show that IBDV uses a previously undescribed VP5-dependent non-lytic egress mechanism significantly enhancing the virus dissemination speed. Ultrastructural analyses revealed that newly assembled IBDV virions associate to a vesicular network apparently facilitating their trafficking from virus assembly factories to the extracellular milieu, and that this association requires the expression of the VP5 polypeptide.

Gga-miR-2127 downregulates the translation of chicken p53 and attenuates chp53-mediated innate immune response against IBDV infection

Infectious bursal disease (IBD) is characterized by the immune suppression of infected birds. The molecular mechanism by which IBD virus (IBDV) suppresses the host immune system remains to be elucidated. The tumor suppressor protein p53 can inhibit the replication of various viruses, but its effect on IBDV remains unknown. This study established an in vitro infection model based on DF-1 cells (chicken embryo fibroblast cell line) to investigate the antiviral effects of chicken p53 (chp53) on IBDV infection. The expression level and activity of chp53 remarkably increased in IBDV-infected DF-1 cells. The overexpression of chp53 inhibited IBDV replication and upregulated the expression of multiple chicken antiviral innate immunity genes (IPS-1, IRF3, PKR, OAS, and Mx), whereas the suppression of chp53 led to the opposite effect. This result indicates that chp53 activates the antiviral innate immune response of chickens to IBDV infection. Bioinformatics analysis and dual-luciferase reporter assay showed that gga-miR-2127 targeted the 3′UTR of chp53. qRT-PCR and western blot revealed that gga-miR-2127 overexpression in DF-1 cells not only downregulated the expression levels of chp53 and of the antiviral innate immunity genes in chickens but also promoted IBDV replication. Our results suggest that gga-miR-2127 downregulates chp53 mRNA translation by targeting its 3′UTR and attenuates chp53-mediated antiviral innate immune response against IBDV.

Eukaryotic translational initiation factor 4AII reduces the replication of infectious bursal disease virus by inhibiting VP1 polymerase activity

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although an interaction between eukaryotic translational initiation factor 4AII (eIF4AII) of the host and viral protein 1 (VP1), the RNA-dependent RNA polymerase (RdRp) of IBDV, has been established, the underlying effects of this interaction on IBDV and the molecular mechanism remain unclear. We here report that interaction of the host eIF4AII with VP1 inhibits the RNA polymerase activity of IBDV to reduce its replication in host cells. We found that ectopically expressed eIF4AII markedly inhibited IBDV growth in DF1 cells, and knockdown of eIF4AII by small interfering RNA significantly enhanced viral replication in CEF cells. Furthermore, IBDV infection led to an increase in host eIF4AII expression, suggesting a feedback mechanism between the host and virus infection both in vitro and in vivo, which further confirmed the involvement of the host eIF4AII in the IBDV life cycle. Thus, via the interaction with VP1, eIF4AII plays a critical role in the IBDV life cycle, by inhibiting viral RNA polymerase activity, leading to a reduction of IBDV replication in cells.

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus

Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 107 pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 105 egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases.

Analysis of the function of D279N mutation of VP2 of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is responsible for the highly contagious infectious bursal disease of chickens. Further understanding the gene-function is necessary to design the tailored vaccine. The amino acid residue 279, located on strand PF of VP2, is one of the three residues that have been reported to be involved in cell-tropism but with some inconsistency. In this study, to further clarify the amino acids involved in the cell tropism of IBDV, a series of mutations about residue 279 were introduced into the VP2 of vvIBDV Gx strain. With the reverse genetic system, we found single mutation of D279N, double mutations of D279N/A284T or Q253H/D279N were not enough to adapt IBDV to chicken embryo fibroblast (CEF) cell. To evaluate whether residue 279 could influence the replication and virulence of IBDV, the virus rGxHT-279 with three mutations (Q253H/D279N/A284T) was rescued and evaluated. Results showed that the mutation of residue 279 in VP2 had no efficient effects on both the replication efficiency in vitro and the virulence to SPF chickens of IBDV. In summary, the results demonstrated that residue 279 of VP2 did not contribute efficiently to cell tropism, replication efficiency, and virulence of IBDV at least in some strains. These findings provided further information for understanding the gene function of IBDV.

Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus

VP3 protein is a structural protein which plays important roles in the virus assembly and the inhibition of antiviral innate immunity of infectious bursal disease virus (IBDV). To explore the potential roles of VP3 in the interplay of IBDV with the host cell, an immunoprecipitation (IP)-coupled mass spectra (MS) screening was performed and the host cellular ribosomal protein L4 (RPL4) was identified as a putative interacting partner of VP3 protein. The interaction of RPL4 with VP3 was further confirmed by co-immunoprecipitation (co-IP) and their colocalization in DF1 cells were observed by confocal microscopy. In addition, knockdown of RPL4 in DF1 cells resulted in reductions of the viral protein pVP2 expression and the virus titers, which reveals a significant role of RPL4 in IBDV replication. Taken together, we indicated for the first time that ribosomal protein L4 (RPL4) was an interacting partner of VP3 and involved in the modulation of IBDV replication. The present study contributes to further understanding the pathogenic mechanism of IBDV.

Gga-miR-9* inhibits IFN production in antiviral innate immunity by targeting interferon regulatory factor 2 to promote IBDV replication

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to the repertoire of host–pathogen interactions during viral infections. In the current study, miRNA analysis showed that a panel of microRNAs, including gga-miR-9*, were markedly upregulated in specific-pathogen-free (SPF) chickens upon infection with infectious bursal disease virus (IBDV); however, the biological function of gga-miR-9* during viral infection remains unknown. Using a TCID50 assay, it was found that ectopic expression of gga-miR-9* significantly promoted IBDV replication. In turn, gga-miR-9* negatively regulated IBDV-triggered type I IFN production, thus promoting IBDV replication in DF-1 cells. Bioinformatics analysis indicates that the 3′ untranslated region (UTR) of interferon regulatory factor 2 (IRF2) has two putative binding sites for gga-miR-9*. Targeting of IRF2 3′UTR by gga-miR-9* was determined by luciferase assay. Functional overexpression of gga-miR-9*, using gga-miR-9* mimics, inhibited IRF2 mRNA and protein expression. Transfection of the gga-miR-9* inhibitor abolished the suppression of IRF2 protein expression. Furthermore, IRF2 knockdown mediated the enhancing effect of gga-miR-9* on the type I IFN-mediated antiviral response. These findings indicate that inducible gga-miR-9* feedback negatively regulates the host antiviral innate immune response by suppressing type I IFN production via targeting IRF2.

Chondroitin sulfate N-acetylgalactosaminyltransferase-2 contributes to the replication of infectious bursal disease virus via interaction with the capsid protein VP2.

Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2.

Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus.

Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.

Oral administration of Allium sativum extract protects against infectious bursal disease in chickens

Garlic (Allium sativum, Liliaceae) has been safely used for more than 5000 years, and research on garlic extract is rapidly increasing because of its multiple biological functions. The in vivo effects of oral administration of garlic mixture (GM, water-soluble extract) on infectious bursal disease virus (IBDV)-infected specific pathogen free male white leghorn chicken were examined through histopathological, immunohistochemical, and Western blot analyses, and enzyme-linked immunosorbent assay. The results confirmed the protective effects of oral administration of 5 mg$kg BW GM (Group GM1) on bursal lesions after IBDV infection. In particular, protein expression of IBDV in the bursa decreased in Group GM1, indicating that GM administration decreased IBDV replication in the bursa. Furthermore, immunoglobulin Mand A-bearing B lymphocytes significantly increased 7 days post infection in bursae in Group GM1 (P< 0.01), suggesting that the oral administration of 5 mg$kg GM offers moderate protection against B cell destruction after IBDV infection. During infection, the concentration of bursal interferon gamma (IFN-g) increased and peaked in Group GM1 earlier than in Group T (IBDV-exposed), demonstrating that GM administration prompted the production of IFN-g to protect against IBDV infection.

Rescue of Infectious Birnavirus from Recombinant Ribonucleoprotein Complexes

Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed, results presented here also support the previously proposed evolutionary connection between birnaviruses and positive-strand single-stranded RNA viruses.

Growth and Replication of Infectious Bursal Disease Virus in the DF-1 Cell Line and Chicken Embryo Fibroblasts

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. To determine a suitable cell line for IBDV infection, replication, and growth kinetics of the virus, DF-1 cells and chicken embryo fibroblasts (CEF) were used. The population doubling per day (Pd/D) was found to be higher in DF-1 as compared to CEF cells. A suitable time of infection (TOI) was established for increased production of virus and greater infectivity titers. The DF-1 and CEF cells were found to be susceptible to infection by producing marked cytopathic effects (CPEs), and the growth curves of IBDV in DF-1 and CEF cells were evaluated by infectivity assay using tissue culture infectious dose (TCID50). The cytopathic effects of the virus in DF-1 and CEF cells were found to be similar, but higher viral titers were detected in the DF-1 cells as compared to CEF. Thus the DF-1 cell line had a higher growth potential and infectivity, which will be of advantage in vaccine production.

Overexpression of microRNA gga-miR-21 in chicken fibroblasts suppresses replication of infectious bursal disease virus through inhibiting VP1 translation

Previously we have identified a series of cellular miRNA molecules up- or down-regulated in infectious bursal disease virus (IBDV) infected chicken embryo fibroblasts and Bursa of Fabricius with gene microarray analysis. Here we studied in detail a relatively well studied miRNA, gga-miR-21, for better understanding miRNAs involvement in IBDV-host interactions. Chicken pri-gga-miRNA-21 and a control miRNA Caenorhabditis elegans pri-cel-lin-4 gene were cloned into a lentiviral vector, respectively. The resulting recombinant lentiviruses were used to infect chicken fibroblast cell line DF-1, and two stable cell lines, DF-miR-21 (overexpressing gga-miR-21) and DF-lin-4 (overexpressing cel-lin-4), were selected. Replication of IBDV in DF-miR-21, DF-lin-4 and DF-1 cells were compared and molecular mechanism of IBDV replication alteration was explored using bioinformatics, reporter gene system, qRT-PCR and Western blot analysis. IBDV replication was markedly lower in DF-miR-21 than in DF-lin-4 or DF-1 cells. A gga-miR-21 target sequence was identified within IBDV VP1 gene (1713–1734bp). Fusion of a 520nt long partial IBDV VP1 gene containing the target with a luciferase gene resulted in significantly lower transient luciferase activity in DF-miR-21 cells as compared to that in DF-lin-4 or DF-1 cell. Following IBDV infection of the cell lines, VP1 protein level in DF-miR-21 cells was dramatically lower than that in DF-lin-4 or DF-1 cells but VP1 mRNA level was not different. The finding indicated that gga-miR-21 could suppress IBDV replication through down regulating IBDV VP1 expression at translational level.

Mutations of Residues 249 and 256 in VP2 Are Involved in the Replication and Virulence of Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. Although the PDE and PFG domains of the capsid protein VP2 contribute significantly to virulence and fitness, the detailed molecular basis for the pathogenicity of IBDV is still not fully understood. Because residues 253 and 284 of VP2 are not the sole determinants of virulence, we hypothesized that other residues involved in virulence and fitness might exist in the PDE and PFG domains of VP2. To test this, five amino acid changes selected by sequence comparison of the PDE and PFG domains of VP2 were introduced individually using a reverse genetics system into the virulent strain (rGx-F9VP2). Then reverse mutations of the selected residues 249 and 256 were introduced individually into the attenuated strain (rGt). Seven modified viruses were generated and evaluated in vitro (CEF cells) and in vivo (SPF chicken). For residue 249, Q249R could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while R249Q could reduce in vitro and elevate in vivo the replication of rGt; meanwhile Q249R reduced the virulence of rGx-F9VP2 while R249Q increased the virulence of rGt, which indicated that residue 249 significantly contributed to the replication and virulence of IBDV. For residue 256, I256V could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while V256I could reduce in vitro but didn’t change in vivo the replication of rGt; although V256I didn’t increase the virulence of rGt, I256V obviously reduced the virulence of virulent IBDV. The present results demonstrate for the first time, to different extent, residues 249 and 256 of VP2 are involved in the replication efficiency and virulence of IBDV; this is not only beneficial to further understanding of pathogenic mechanism but also to the design of newly tailored vaccines against IBDV.

The endosomal pathway and the Golgi complex are involved in the infectious bursal disease virus life cycle.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.