Molecular Interactions of the Infectious Bursal Disease Virus Proteins

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae (Dobos et al., 1979), is the causative agent of a highly contagious immunosuppressive disease in young chickens. IBDV multiplies rapidly in developing B lymphocytes in the bursa of Fabricius, leading to immunosuppression. This increases susceptibility to infections with opportunistic pathogens and reduces the growth rate of surviving animals. IBDV is a double-stranded RNA (dsRNA) virus. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shelled capsid that is composed of VP2 and VP3. The other (non-structural) viral proteins of IBDV are VP4, a protease, and VP5, which is not essential for viral replication in vitro but important for virus-induced pathogenicity . Viral proteins generally function by interactions with viral and/or host cell proteins. Information about these interactions is thus essential for understanding the infection process. The aim of this thesis therefore was to initiate making the inventory of interactions essential to the IBDV life cycle. To this end, we employed the then recently developed yeast two-hybrid system. First, we focussed on the homo- and heteromeric interactions of the known viral proteins. A heterologous interaction between VP1 and VP3, and homologous interactions of VP2, VP3, VP4, VP5, and possibly of VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. Then, we identified the domains responsible for these interactions. The VP1 interacting domain of VP3 was mapped to the extreme carboxy-terminal domain of the polypeptide. This interaction appeared to be crucial for the production of infectious progeny. These investigations also revealed that VP3 additionally binds to both the viral dsRNA segments. The mapping of the individual contact sites on the self-interacting viral proteins VP2, VP3, VP4 and VP5 revealed that VP2 possesses multiple interaction domains, consistent with available structural information about this external capsid protein. VP3-VP3 interactions were mapped to the amino-terminal part of the polypeptide. No interaction sites could be assigned to the VP4 protein; any deletion applied abolished its self-association. And for VP5, one interaction domain was detected in its central, most hydrophobic region, supporting the idea that this virulence determinant may function as a membrane pore-forming protein in infected cells. Finally, we performed a yeast two-hybrid search for candidate cellular proteins of the bursa of Fabricius interacting with VP1, VP2, VP3 and VP5. We found that several host cell proteins are able to form complexes with the viral proteins of IBDV in yeast cells. A specific interaction of VP1 with the carboxy-terminal domain of eukaryotic initiation factor 4AII (eIF4AII), a key component in the initiation of eukaryotic translation of both capped and uncapped mRNAs, was confirmed by co-immunoprecipitation. The biological relevance of the potential VPg-eIF4AII interaction is discussed. It is now clear that VP3 is the key organizer of the birnavirus structure as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1 and the two genomic dsRNAs. Different domains in the protein are responsible for these different interactions. The association of eIF4AII with IBDV VP1 will require future studies to confirm and establish the functional significance of this interaction for viral multiplication

Protective Efficacy of Newcastle Disease Virus Expressing Soluble Trimeric Hemagglutinin against Highly Pathogenic H5N1 Influenza in Chickens and Mice

Background: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. Methodology/Principal Findings: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5 3). A single intramuscular immunization with NDV-sH5 3 or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5 3 was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5 3 was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. Conclusions/Significance: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles

In vivo interactions between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA dependent polymerase VP1

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent polymerase, VP1

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome

Dose response effects of avian influenza (H7N7) vaccination of chickens: Serology, clinical protection and reduction of virus excretion

Knowledge of the relation between the antigen content of inactivated avian influenza (Al) vaccines, the serological response after vaccination and protection of vaccinated animals is important for the choice of optimal vaccines and vaccination regimes as well as for the assessment of criteria for the licensing of new Al-vaccines. We studied this relation in a dose response study using inactivated H7N7 avian influenza vaccines with varying antigen content. The serological response depended on the antigen content of the vaccines. Anti-Al antibodies were detected most frequently with ELISA, followed by the virus neutralisation test and the haemagglutination inhibition (HI) assay. Chickens with measurable HI-antibody titers, using homologous H7N7 antigen. were all protected against clinical disease after challenge with highly pathogenic A/chicken/Netherlands/621557/03 H7N7 virus. However, in these chickens high levels of virus could still be present on days 2-4 after challenge. The reduction of virus titers after challenge, depended on the antigen content of the vaccines as well as on the serum antibody titers. While 10 haemagglutinating units (HAU), equivalent to 0.8 mu g haemagglutinin (HA) protein, per vaccine dose was sufficient for prevention of clinical disease, 128 HAU (9 mu g HA) per dose was required for reduction of virus titers in all chickens to 10(3) egg-infectious dose 50% (EID50) or less. In order to reduce virus titers below 10(3) EID50 per swab a HI-antibody titer of 64 was required. After use of the vaccine with the highest antigen content, challenge still induced a booster of antibody titers which is indicative of replication of challenge virus

Dose response effects of avian influenza (H7N7) vaccination of chickens: Serology, clinical protection and reduction of virus excretion

Knowledge of the relation between the antigen content of inactivated avian influenza (Al) vaccines, the serological response after vaccination and protection of vaccinated animals is important for the choice of optimal vaccines and vaccination regimes as well as for the assessment of criteria for the licensing of new Al-vaccines. We studied this relation in a dose response study using inactivated H7N7 avian influenza vaccines with varying antigen content. The serological response depended on the antigen content of the vaccines. Anti-Al antibodies were detected most frequently with ELISA, followed by the virus neutralisation test and the haemagglutination inhibition (HI) assay. Chickens with measurable HI-antibody titers, using homologous H7N7 antigen. were all protected against clinical disease after challenge with highly pathogenic A/chicken/Netherlands/621557/03 H7N7 virus. However, in these chickens high levels of virus could still be present on days 2-4 after challenge. The reduction of virus titers after challenge, depended on the antigen content of the vaccines as well as on the serum antibody titers. While 10 haemagglutinating units (HAU), equivalent to 0.8 mu g haemagglutinin (HA) protein, per vaccine dose was sufficient for prevention of clinical disease, 128 HAU (9 mu g HA) per dose was required for reduction of virus titers in all chickens to 10(3) egg-infectious dose 50% (EID50) or less. In order to reduce virus titers below 10(3) EID50 per swab a HI-antibody titer of 64 was required. After use of the vaccine with the highest antigen content, challenge still induced a booster of antibody titers which is indicative of replication of challenge virus

VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed

Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for bluetongue

Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal (DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine, but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with field virus, horizontal spread by vectors and vertical transmission. The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype specific which is dependent on the immunodominant and serotype determining VP2 protein. The BT DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We here established an NS3-specific competitive ELISA (NS3 cELISA) as serological DIVA test accompanying BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety of sera. The NS3 cELISA displayed a high sensitivity and specificity similar to the commercially available VP7-specific cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein