Patterns of RNA Editing in Newcastle Disease Virus Infections.

The expression of accessory non-structural proteins V and W in Newcastle disease virus (NDV) infections depends on RNA editing. These proteins are derived from frameshifts of the sequence coding for the P protein via co-transcriptional insertion of one or two guanines in the mRNA. However, a larger number of guanines can be inserted with lower frequencies. We analysed data from deep RNA sequencing of samples from in vitro and in vivo NDV infections to uncover the patterns of mRNA editing in NDV. The distribution of insertions is well described by a simple Markov model of polymerase stuttering, providing strong quantitative confirmation of the molecular process hypothesised by Kolakofsky and collaborators three decades ago. Our results suggest that the probability that the NDV polymerase would stutter is about 0.45 initially, and 0.3 for further subsequent insertions. The latter probability is approximately independent of the number of previous insertions, the host cell, and viral strain. However, in LaSota infections, we also observe deviations from the predicted V/W ratio of about 3:1 according to this model, which could be attributed to deviations from this stuttering model or to further mechanisms downregulating the abundance of W protein.

2ß-3,4-Unsaturated sialic acid derivatives: Synthesis optimization, and biological evaluation as Newcastle disease virus hemagglutinin-neuraminidase inhibitors.

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.

Construction and expression of a single-chain variable fragment antibody against chicken DEC 205 for targeting the bacterial expressed hemagglutinin-neuraminidase of Newcastle disease virus.

Targeting antigens to endocytic receptors on the surface of dendritic cells is a new strategy for increasing the adaptive immune response. The objective of the current study was the construction and bacterial expression of a recombinant antibody single-chain fragment variable (ScFv) directed against chicken DEC 205, an endocytic receptor, for use in the genetic fusion of antigens. In particular, we use as antigen the hemagglutinin-neuraminidase (HN) of Newcastle disease virus. Our results show that inoculation of chickens with HN genetically fused to the ScFv anti-DEC 205 induced an evidently higher immune response against HN, in contrast to inoculation with unconjugated HN. In addition, neutralizing antibodies against Newcastle disease virus were detected only in the serum from chickens immunized with HN fused to ScFv anti-DEC 205. Inoculated fused antigens to ScFv against endocytic receptor DEC 205 resulted in a greater antibody-specific anti-HN production compared with antigens applied alone. The results of this study show that the strategy described here has the potential to be used in the development of more effective vaccines against infectious diseases in chickens.

Recombinant Newcastle disease virus (NDV) La Sota expressing the haemagglutinin-neuraminidase protein of genotype VII NDV shows improved protection efficacy against NDV challenge.

Intensive vaccination strategies against Newcastle disease (ND) have been implemented in many countries for a long time, but ND outbreaks still occur frequently, with most isolates belonging to genotype VII of Newcastle disease virus (NDV). Many researchers have revealed that vaccines closely matched to epidemic viruses provide better protection. Therefore, using a previously established reverse genetics system, we generated a recombinant NDV vaccine strain (rLa Sota-HN) based on the La Sota vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of genotype VII NDV. The pathogenicity of the recombinant virus was confirmed by the mean death time in 9-day-old specific-pathogen-free embryonated chicken eggs and the intracerebral pathogenicity index in 1-day-old specific-pathogen-free chickens. Subsequently, 1-day-old chickens were immunized with commercial vaccine La Sota and recombinant virus rLa Sota-HN and then challenged with virulent genotype VII NDV strain. The results indicated that recombinant virus rLa Sota-HN provided increased protection of vaccinated chickens from morbidity and mortality, and inhibited the shedding of virulent virus after challenging with genotype VII virus, compared with the conventional vaccine La Sota. Our findings indicated that rLa Sota-HN is a promising vaccine candidate to improve the protection efficiency against ND in chickens, thereby preventing frequent outbreaks of this disease.

Hemagglutinin-Neuraminidase and fusion genes are determinants of NDV thermostability.

Newcastle disease (ND) caused by infections with virulent strains of Newcastle disease virus (NDV) continues to be a threat for poultry industry worldwide. The prospect of developing a thermostable and effective NDV vaccine is still highly desirable. To investigate the determinants of thermostability in NDV, we generated recombinant NDV strains by exchanging viral hemagglutinin-neuraminidase (HN) gene or by mutating the fusion (F) gene. The results showed that the HN and F protein were both determinants of NDV thermostability. With increased thermostability, the HN protein-chimeric virus showed significantly reduced neuraminidase and hemadsorption activities, but its hemolytic activity was retained. We also found that changing the amino acid in the F protein cleavage sites, affected the thermostability as well as the pathogenicity and fusogenic capacity of the virus. Taken together, our results suggest that HN and F proteins both contribute to the thermostability of NDV, and other viral biological activities change as the thermostability of the virus changes. These findings should be of benefit to the development of a thermostable and efficacious NDV vaccine.

Antitumor effect of the Newcastle disease viral hemagglutinin-neuraminidase gene is expressed through an oncolytic adenovirus effect in osteosarcoma cells.

Newcastle disease virus (NDV) can specifically kill cancer cells and has less toxicity to normal cells. The hemagglutinin-neuraminidase (HN) protein is an important structural protein in NDV pathogenesis and has been postulated as a promising candidate for antitumor therapy. The aim of this study was to investigate the anticancer potential of recombinant adenovirus Ad-HN-PEG3p-E1a. An MTS assay was performed to determine viral proliferation after viral infection, the data showed that the proliferation ability of osteosarcoma cells decreased, whereas there was no significant change in normal hepatic cells. DAPI and Annexin V experiments showed that osteosarcoma cells were killed because of apoptosis, active oxygen content, and augmented mitochondrial membrane potential loss. Caspase Activity Assay Kits were used to detect the caspase-3 activities of the treated OS-732 for increased expression. Western blot analysis showed that cytochrome C increased significantly and apoptosis of the virus was confirmed in tumor cells. In-vivo experiments show that NDV has an inhibitory effect on tumor growth. The recombinant adenovirus, which is composed of a HN protein and progressive increment promoter PEG3p, could inhibit the growth of OS-732 and promote the apoptosis of tumor cells. However, there was no clear relationship with normal cell (L02) apoptosis.

Synonymous codon usage of genes in polymerase complex of Newcastle disease virus.

Newcastle disease virus (NDV) is pathogenic to both avian and non-avian species but extensively finds poultry as its primary host and causes heavy economic losses in the poultry industry. In this study, a total of 186 polymerase complex comprising of nucleoprotein (N), phosphoprotein (P), and large polymerase (L) genes of NDV was analyzed for synonymous codon usage. The relative synonymous codon usage and effective number of codons (ENC) values were used to estimate codon usage variation in each gene. Correspondence analysis (COA) was used to study the major trend in codon usage variation. Analyzing the ENC plot values against GC3s (at synonymous third codon position) we concluded that mutational pressure was the main factor determining codon usage bias than translational selection in NDV N, P, and L genes. Moreover, correlation analysis indicated, that aromaticity of N, P, and L genes also influenced the codon usage variation. The varied distribution of pathotypes for N, P, and L gene clearly suggests that change in codon usage for NDV is pathotype specific. The codon usage preference similarity in N, P, and L gene might be detrimental for polymerase complex functioning. The study represents a comprehensive analysis to date of N, P, and L genes codon usage pattern of NDV and provides a basic understanding of the mechanisms for codon usage bias.

Neuraminidase as an enzymatic marker for detecting airborne Influenza virus and other viruses.

Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis µ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

Contribution of HN protein length diversity to Newcastle disease virus virulence, replication and biological activities.

To evaluate the contribution of length diversity in the hemagglutinin-neuraminidase (HN) protein to the pathogenicity, replication and biological characteristics of Newcastle disease virus (NDV), we used reverse genetics to generate a series of recombinant NDVs containing truncated or extended HN proteins based on an infectious clone of genotype VII NDV (SG10 strain). The mean death times and intracerebral pathogenicity indices of these viruses showed that the different length mutations in the HN protein did not alter the virulence of NDV. In vitro studies of recombinant NDVs containing truncated or extended HN proteins revealed that the extension of HN protein increased its hemagglutination titer, receptor-binding ability and impaired its neuraminidase activity, fusogenic activity and replication ability. Furthermore, the hemadsorption, neuraminidase and fusogenic promotion activities at the protein level were consistent with those of viral level. Taken together, our results demonstrate that the HN biological activities affected by the C-terminal extension are associated with NDV replication but not the virulence.

Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

Comparative analysis of sialidase protein in velogenic and lentogenic strains of Newcastle disease virus.

The sialidase protein is a  major part of hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV), which is an important multifunctional envelope protein. This protein plays key roles in virus attachment to cells and virus-cell fusion. In this study, we compared the sialidase protein of Iranian virulent velogenic field strains with that of avirulent lentogenic strains. Six of seventeen variations in amino acid 395, 523, 550 432, 479 and 540 were observed near the catalytic and glycosylation sites in the sialidase protein. The obtained results showed fundamental differences in various biological parameters such as post-translational modification, antigenic index and electrostatic potential of tertiary structure of the sialidase protein. We suggest these six amino acids might play an effective role in the pathogenesis of NDV.

Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process.

Newcastle disease virus (NDV), an avain paramyxovirus, has been assigned to the genus Avulavirus within the family Paramyxoviridae. It causes Newcastle disease (ND) that is a highly contagious and fatal viral disease affecting poultry and most species of birds. The hemagglutinin-neuraminidase (HN) protein of NDV has multiple functions including mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities affecting the process of viral attachment, entry, replication and dissemination. Fusion ability of the NDV was highly correlated to its virulence. Mutations in the HN globular head and headless HN of NDV were constructed to determinate the impact of highly conserved amino acids in the globular head of paramyxovirus HN proteins and the roles of the stalk region of HN in the fusion process. It was found that the interaction between F and HN mutants E401A, G402A, G468A, V469A, Y526A, and T527A was equal to that in F and wt HN. The mutations of G402A, G468A, V469A, and T527A had various effects on cell fusion promotion, receptor binding ability, and NA activity, but the membrane merging rate was comparable to wt HN. The elimination of hemadsorption ability and NA activity of E401A and Y526A resulted in the loss of the fusion promotion function of HN. The conclusion was that receptor binding and NA had a common active site and E401 and Y526 amino acids were essential for virus attachment, entry, and dissemination. In addition, G468A mutation made different contributions to HAD and NA, which indicated that G468 was one of the potential key amino acids in switching the two functions between receptor binding and sialic acid destruction of HN. It was also proven that the headless HN of NDV could promote the fusion event mediated by F. Thus, it revealed a novel mechanism in F activation of NDV.

A novel method for detection of Newcastle disease virus with a fluorescent sialidase substrate.

Newcastle disease virus (NDV), belonging to the family Paramixoviridae, causes respiratory and neuronal symptoms in almost all birds. NDV has haemagglutinin-neuraminidase (HN) glycoprotein possessing sialidase activity. HN glycoprotein is highly expressed on the surface of NDV-infected cells, resulting in much higher sialidase activity in NDV-infected cells than in non-infected cells. It was reported that mouse and human cancer cells up-regulating sialidase expression were histochemically stained with a fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), which deposits water-insoluble fluorescent compound BTP3 on locations of sialidase activity. By using the BTP3-Neu5Ac assay, we showed that NDV-infected cells and HN gene-expressing cells could be simply detected at room temperature after only 5min. Infection of the cells with the virus resulted in apparent green fluorescence, which disappeared with addition of a sialidase inhibitor. Cells that were stained in the BTP3-Neu5Ac assay were immunostained with an anti-NDV antibody. Moreover, BTP3-Neu5Ac staining was applied to a virus overlay binding assay with NDV particles. NDV-bound protein bands on guinea pig red blood cells were easily and rapidly detected by the BTP3-Neu5Ac assay after Western blotting. BTP3-Neu5Ac offers an easy and rapid protocol for fluorescent staining of NDV and virus-infected cells without antibodies.

Role of C596 in the C-terminal extension of the haemagglutinin-neuraminidase protein in replication and pathogenicity of a highly virulent Indonesian strain of Newcastle disease virus.

We modified the haemagglutinin-neuraminidase (HN) glycoprotein of the virulent Newcastle disease virus (NDV) strain Banjarmasin/010/10 (Ban/010) by adding C-terminal extensions similar to those found in certain avirulent NDV strains. Extension of the 571 aa wt Ban/010 HN protein to 577 and 616 aa by removal of one or two translational stop codons moderately reduced HN function and viral pathogenicity in 1-day-old and 3-week-old chickens. Substantially greater reductions were achieved by altering the 616 aa form by introducing a R596C mutation or by replacing the C-terminal extension with that of avirulent strain Ulster, which naturally contains the amino acid 596C. These results showed that extension of the C terminus of HN reduces NDV pathogenicity, and that this effect is substantially increased by the presence of 596C. These results indicate that this attenuating mechanism in avirulent strains such as Ulster can be applied directly to a highly virulent strain recently in circulation.

'a'-Position-mutated and G4-mutated hemagglutinin-neuraminidase proteins of Newcastle disease virus impair fusion and hemagglutinin-neuraminidase-fusion interaction by different mechanisms.

OBJECTIVES: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. METHODS: We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. RESULTS: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. CONCLUSIONS: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures.

Structure of the ulster strain newcastle disease virus hemagglutinin-neuraminidase reveals auto-inhibitory interactions associated with low virulence.

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase ß-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN0 and associated reduced virulence.

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.

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.

Positive selection in the hemagglutinin-neuraminidase gene of Newcastle disease virus and its effect on vaccine efficacy.

BACKGROUND: To investigate the relationship between the selective pressure and the sequence variation of the hemagglutinin-neuraminidase (HN) protein, we performed the positive selection analysis by estimating the ratio of non-synonymous to synonymous substitutions with 132 complete HN gene sequences of Newcastle disease viruses (NDVs) isolated in China. RESULTS: The PAML software applying a maximum likelihood method was used for the analysis and three sites (residues 266, 347 and 540) in the HN protein were identified as being under positive selection. Codon 347 was located exactly in a recognized antigenic determinant (residues 345-353) and codon 266 in a predicted linear B-cell epitope. Substitutions at codon 540 contributed to the N-linked glycosylation potential of residue 538. To further evaluate the effect of positively selected sites on the vaccine efficacy, we constructed two recombinant fowlpox viruses rFPV-JS6HN and rFPV-LaSHN, expressing the HN proteins from a genotype VII field isolate Go/JS6/05 (with A266, K347 and A540) and vaccine strain La Sota (with V266, E347 and T540), respectively. Two groups of SPF chickens, 18 each, were vaccinated with the two recombinant fowlpox viruses and challenged by Go/JS6/05 at 3 weeks post-immunization. The results showed that rFPV-JS6HN could elicit more effective immunity against the prevalent virus infection than rFPV-LaSHN in terms of reducing virus shedding. CONCLUSIONS: The analysis of positively selected codons and their effect on the vaccine efficacy indicated that the selective pressure on the HN protein can induce antigenic variation, and new vaccine to control the current ND epidemics should be developed.

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.