Newcastle Disease Virus Hemagglutinin-neuraminidase Research Articles

The Second Receptor Binding Site of the Globular Head of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Activates the Stalk of Multiple Paramyxovirus Receptor Binding Proteins To Trigger Fusion

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN's globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN's site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.

Role of the Two Sialic Acid Binding Sites on the Newcastle Disease Virus HN Protein in Triggering the Interaction with the F Protein Required for the Promotion of Fusion

Newcastle disease virus (NDV)-induced membrane fusion requires an interaction between the hemagglutinin-neuraminidase (HN) attachment and the fusion (F) proteins, triggered by HN's binding to receptors. NDV HN has two sialic acid binding sites: site I, which also mediates neuraminidase activity, and site II, which straddles the membrane-distal end of the dimer interface. By characterizing the effect on receptor binding avidity and F-interactive capability of HN dimer interface mutations, we present evidence consistent with (i) receptor engagement by site I triggering the interaction with F and (ii) site II functioning to maintain high-avidity receptor binding during the fusion process.

Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

Roles of the Fusion and Hemagglutinin-Neuraminidase Proteins in Replication, Tropism, and Pathogenicity of Avian Paramyxoviruses

Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of APMV-2 with only a minimal attenuating effect. These results demonstrate that the F and HN ectodomains together determine the cell fusion, tropism, and virulence phenotypes of NDV and APMV-2 and that the regions of HN that are critical to replication and the species-specific phenotypes include the cytoplasmic tail and stalk domain but not the globular head domain.

Triggering of the Newcastle Disease Virus Fusion Protein by a Chimeric Attachment Protein That Binds to Nipah Virus Receptors

The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding.

Enhanced Reliability of Avian Influenza Virus (AIV) and Newcastle Disease Virus (NDV) Identification Using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS)

In-solution enzymatic and nonenzymatic digestion methods have been successfully implemented in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based virus identification, extending to typing/subtyping of deadly influenza viruses. However, these methods are inefficient in obtaining more precise information on surface proteins of myxovirus particles, not only the hemagglutinin and neuraminidase of influenza virus but also the hemagglutinin-neuraminidase of Newcastle disease virus (NDV). Imbalances in viral protein composition cause ion suppression of tryptic fragments from low-abundant target proteins (surface proteins), adversely affecting reproducibility of mass spectra. Additionally, the coexistence of tryptic peptides from several proteins requires sophisticated statistical solutions for precise result interpretations. To circumvent these, we apply detergent-based (gel-free) partitioning of whole viruses into soluble surface proteins and insoluble virus materials, using differential centrifugation. MALDI-TOF or MALDI-TOF/TOF MS was applied to analyze tryptic peptides from separated viral proteins. In this study, we achieved type/subtype of avian influenza virus (AIV) within 5 h, based on 4 major proteins, by significantly reducing ion suppression and signal overlap from various protein sources. Hence, our approach can both yield dependable results and allow Web-based search engines to be directly employed, obviating the need for additional statistical strategy. Additionally, we demonstrate the utility of the method using NDV.

The Transmembrane Domain Sequence Affects the Structure and Function of the Newcastle Disease Virus Fusion Protein

The role of specific sequences in the transmembrane (TM) domain of Newcastle disease virus (NDV) fusion (F) protein in the structure and function of this protein was assessed by replacing this domain with the F protein TM domains from two other paramyxoviruses, Sendai virus (SV) and measles virus (MV), or the TM domain of the unrelated glycoprotein (G) of vesicular stomatitis virus (VSV). Mutant proteins with the SV or MV F protein TM domains were expressed, transported to cell surfaces, and proteolytically cleaved at levels comparable to that of the wild-type protein, while mutant proteins with the VSV G protein TM domain were less efficiently expressed on cell surfaces and proteolytically cleaved. All mutant proteins were defective in all steps of membrane fusion, including hemifusion. In contrast to the wild-type protein, the mutant proteins did not form detectable complexes with the NDV hemagglutinin-neuraminidase (HN) protein. As determined by binding of conformation-sensitive antibodies, the conformations of the ectodomains of the mutant proteins were altered. These results show that the specific sequence of the TM domain of the NDV F protein is important for the conformation of the preactivation form of the ectodomain, the interactions of the protein with HN protein, and fusion activity.

Antitumor vaccination by Newcastle Disease Virus Hemagglutinin–Neuraminidase plasmid DNA application: Changes in tumor microenvironment and activation of innate anti-tumor immunity

A plasmid encoding the Hemagglutinin–Neuraminidase (HN) protein of Newcastle Disease Virus (pHN) was tested for its capacity to stimulate innate anti-tumor activity in tumor-bearing mice. We observed that application of the pHN plasmid at the ear pinna site (i.e.) of mice induces higher levels of systemic interferon-α and reduced tumor growth in the prophylactic mammary carcinoma DA3 tumor model in comparison to application of a control plasmid not encoding the HN protein. Analysis of the tumor microenvironment revealed a significant increase in NK cell infiltration and decrease in infiltration of CD11b+Gr-1high myeloid cells bearing the myeloid-derived suppressor cell (MDSC) phenotype after vaccination with the pHN DNA compared to a control DNA. Finally, innate immunity and partially type I IFN responses were proved important for the reduction of s.c. RMA-S tumor growth after pHN vaccination, as shown with the use of RAG2−/− and RAG2−/−IFNAR1−/− mice.These data demonstrate that triggering innate immunity by pHN application at the ear pinna of mice modulates the immune cell compartment in the tumor microenvironment and reduces tumor growth. This highlights thus the potential adjuvant activity of the HN gene in tumor therapy.

Anti-tumor immunity of Newcastle disease virus HN protein is influenced by differential subcellular targeting

背景与目的新城疫病毒HN蛋白是新城疫病毒产生溶瘤作用的重要免疫原。在前期体外实验基础上，比较定位表达细胞不同部位的HN蛋白体内抗肿瘤免疫作用。方法通过构建荷瘤小鼠，瘤内注射定位表达于细胞不同部位的新城疫病毒HN蛋白，即胞浆型（Cy-HN）、跨膜型（M-HN）、分泌型（Sc-HN）重组真核表达质粒，比较荷瘤小鼠的肿瘤生长速度，脾淋巴细胞增殖反应和细胞毒T细胞活性。结果瘤内注射跨膜型重组真核表达质粒的荷瘤小鼠肿瘤生长缓慢，与瘤内注射胞浆型和分泌型重组真核表达质粒的荷瘤小鼠相比有统计学差异（第18天：P=0.022；第21天：P < 0.01），同时，该组荷瘤小鼠的淋巴细胞增殖反应和细胞毒T细胞活性也较高[M-HN vs Cy-HN, P=0.019; M-HN vs Sc-HN, P=0.043; M-HN vs pcDNA3.1(+), P < 0.01]。结论定位表达于细胞不同部位的HN蛋白体内抗肿瘤免疫作用存在差异，跨膜型HN蛋白的重组DNA质粒能够提高荷瘤小鼠的特异性细胞免疫应答。

Assembly and Biological and Immunological Properties of Newcastle Disease Virus-Like Particles

Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.

The anti-tumor effect of Newcastle disease virus HN protein is influenced by differential subcellular targeting.

Immunotherapy is emerging as a major player in the current standard of care for aggressive cancers such as non-small cell lung cancer (NSCLC). The Newcastle disease virus with its tumor-specific replicative and oncolytic abilities is a promising immunotherapeutic candidate. A DNA vaccine expressing the major immunogenic hemagglutinin-neuraminidase (HN) protein of this virus has shown promising results as an immunotherapeutic agent. In the present study, three different DNA vaccine constructs encoding differentially targeted HN proteins (cytoplasmic or Cy-HN, secreted or Sc-HN and membrane-anchored or M-HN) were generated to evaluate their anti-tumor effect in vitro and in vivo. Although all three DNA constructs elicited an immune response, tumor-bearing mice intratumorally injected with M-HN demonstrated a significantly better anti-tumor effect than those injected with Cy-HN or Sc-HN. We also showed that this anti-tumor effect was mediated by higher lymphocyte proliferative response and CTL activity in mice intratumorally injected with M-HN. The membrane-anchored form of the HN protein appears to be an ideal candidate to develop as an immunotherapeutic agent for NSCLC.

N-Linked Glycan at Residue 523 of Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Masks a Second Receptor-Binding Site

The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second receptor-binding site identified in Newcastle disease virus (NDV) HN (I. A. Alymova, G. Taylor, V. P. Mishin, M. Watanabe, K. G. Murti, K. Boyd, P. Chand, Y. S. Babu, and A. Portner, J. Virol. 82:8400-8410, 2008). Sequence analysis and superposition of the NDV and hPIV-3 HN dimer structures revealed that, similar to what was seen in hPIV-1, the N-linked glycan at residue 523 on hPIV-3 HN may cover a second receptor-binding site. Removal of this N-linked glycosylation site by an Asn-to-Asp substitution at residue 523 (N523D) changed the spectrum of the mutant virus's receptor specificity, delayed its elution from both turkey and chicken red blood cells, reduced mutant sensitivity (by about half) to the selective HN inhibitor BCX 2855 in hemagglutination inhibition tests, and slowed its growth in LLC-MK(2) cells. The neuraminidase activity of the mutant and its sensitivity to BCX 2855 in neuraminidase inhibition assays did not change, indicating that the mutation did not affect the virus's catalytic-binding site and that all observed effects were caused by the exposure of the purported second receptor-binding site. Our data are consistent with the idea that, similar to the case for hPIV-1, the N-linked glycan shields a second receptor-binding site on hPIV-3 HN.

Increase in the Neuraminidase Activity of a Nonpathogenic Newcastle Disease Virus Isolate during Passaging in Chickens

A pathogenic mutant of the Newcastle disease virus (NDV) was previously generated by passaging a non-pathogenic isolate from wild waterfowl. Velogenic mutant 9a5b (IVPI=2.67) contains three amino acid substitutions (128H, 495K and 573stop) in the hemagglutinin-neuraminidase (HN) protein, as compared with nonpathogenic waterfowl isolate 415/91 strain, and two of these (128H and 495K) were introduced after mesogenic 9a3b (IVPI=1.88). To investigate the role of the HN protein in NDV virulence, the function of HN protein such as neuraminidase (NA), Hemadsorption (HAd) and fusion promotion activities was examined by introducing the point mutations observed in passaged mutants into the HN gene cDNAs. In vitro functional assay using mutant protein expression demonstrated that the 128H substitution markedly increases NA activity and 573stop substitution increase NA and HAd activities. On the other hand, 495K substitution had little effect on any activities. These results indicate that a single amino acid substitution (128P to H) in the NDV HN protein affects the neuraminidase activity and is possibly correlated with the virulence.

Identification of a variable epitope on the Newcastle disease virus hemagglutinin–neuraminidase protein

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin–neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345–353) on the HN protein.

Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation, Cell Fusion, and Pathogenicity

To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.

A Y526Q Mutation in the Newcastle Disease Virus HN Protein Reduces Its Functional Activities and Attenuates Virus Replication and Pathogenicity

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.

Engineered Intermonomeric Disulfide Bonds in the Globular Domain of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein: Implications for the Mechanism of Fusion Promotion

The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37 degrees C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.

Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

Avian Adeno-Associated Virus-Based Expression of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein for Poultry Vaccination

The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus member of the family Parvoviridae that has been proved to be useful as a viral vector for gene delivery. The use of AAAV for transgenic expression of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein and its ability to induce immunity in chickens were assessed. Proposed advantages of this system include no interference with maternal antibodies, diminished immune response against the vector, and the ability to accommodate large fragments of genetic information. In this work the generation of recombinant AAAV virions expressing the HN protein (rAAAV-HN) was demonstrated by electron microscopy, immunocytochemistry, and western blot analysis. Serological evidence of HN protein expression after in ovo or intramuscular inoculation of the recombinant virus in specific-pathogen-free chickens was obtained. Serum from rAAAV-HN-vaccinated birds showed a systemic immune response evidenced by NDV-specific enzyme-linked immunosorbent assay and hemagglutination inhibition testing. Positive virus neutralization in embryonated chicken eggs and indirect immunofluorescence detection of NDV infected cells by serum from rAAAV-HN vaccinated birds is also reported. A vaccine-challenge experiment in commercial broiler chickens using a Venezuelan virulent viscerotropic strain of NDV was performed. All unvaccinated controls died within 5 days postchallenge. Protection up to 80% was observed in birds vaccinated in ovo and revaccinated at 7 days of age with the rAAAV-HN. The results demonstrate the feasibility of developing and using an AAAV-based gene delivery system for poultry vaccination.

A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus

The protection of poultry from H5N1 highly pathogenic avian influenza A (HPAI) and Newcastle disease virus (NDV) can be achieved through vaccination, as part of a broader disease control strategy. We have previously generated a recombinant influenza virus expressing, (i) an H5 hemagglutinin protein, modified by the removal of the polybasic cleavage peptide and (ii) the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein in the place of the ectodomain of influenza neuraminidase (Park MS, et al. Proc Natl Acad Sci USA 2006;103(21):8203-8). Here we show this virus is attenuated in primary normal human bronchial epithelial (NHBE) cell culture, and demonstrate protection of C57BL/6 mice from lethal challenge with an H5 HA-containing influenza virus through immunisation with the recombinant virus. In addition, in-ovo vaccination of 18-day-old embryonated chicken eggs provided 90% and 80% protection against highly stringent lethal challenge by NDV and H5N1 virus, respectively. We propose that this virus has potential as a safe in-ovo live, attenuated, bivalent avian influenza and Newcastle disease virus vaccine.