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Newcastle disease is a highly contagious viral infection affecting many species of birds that can spread fast between poultry houses and cause a heavy economic burden on the poultry industry all around the world. Fusion and hemagglutinin-neuraminidase (HN) protein are important in the pathogenesis of the Newcastle disease virus (NDV). The HN protein is a critical viral protein with multiple functions and plays a key role in the formation of the virulence of NDV. Head of HN protein is responsible for receptor binding, neuraminidase activity. This study aimed to investigate the sequence homology of hemagglutinin-neuraminidase of two NDV isolates sampled from infected farms in Iran. The samples were collected from flocks that had been vaccinated by both types of live and killed vaccines for NDV. After isolation of NDV, the viruses were subjected to the polymerase chain reaction (PCR) amplification using two pairs of specific primers designed for the HN gene to amplify the complete HN gene (1730bp). Afterward, the PCR products were sequenced and analyzed by phylogenetic tree construction software. Based on the analysis, substantial sequence homology among Iranian isolates is within the range of 97.1-100%. Moreover, the sequence homology searching revealed a level of similarity between HN sequences of Iranian isolates and the HN sequences from other countries, particularly Asian ones. For instance, a high homology ratio (95.34%) was found between Iranian isolates and the sequences registered on online molecular databases from China. Based on phylogenetic analysis, the NDV isolates belong to the VIId genotype. Finally, it can be concluded that monitoring the circulation of NDVs among poultry and other birds can help to obtain an insight into the evolution of NDVs and control of panzootic viruses in the future.

Background The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization. Methods In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmids and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test. Results Five IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328–342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242–256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope. Conclusion The HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242–256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.

Newcastle disease is a kind of avian infectious disease caused by Newcastle disease virus (NDV). The virulence of NDV is dependent mainly on the fusion (F) protein and hemagglutinin-neuraminidase (HN) protein. The genomes of 2 viruses, NDV-Blackbird and NDV-Dove, are 99.9% similar, while NDV-Blackbird is a velogenic virus, and NDV-Dove is a lentogenic virus. Further analysis revealed that the F proteins of the 2 strains were identical, and only 5 amino acid sites on the HN proteins were inconsistent. Five different HN mutant plasmids were constructed and analyzed in this study. The results showed that the mutation F110L caused a significant increase in fusion-promotion activity caused by an increase in neuraminidase activity. Because of the increase in receptor-binding activity caused by G116R, there was a significant increase in fusion-promotion activity. The mutation G54S resulted in a slight decrease in the fusion-promotion activity caused by a slight decrease in receptor-binding activity. The slight increase in the fusion-promotion activity caused by A469V was associated with a significant increase in neuraminidase activity. Therefore, the amino acids L110 and R116 played a key role in determining the virulence difference between NDV-Blackbird and NDV-Dove, which could lay a foundation for illuminating the virulence differences of NDV strains, as well as the development of attenuated vaccines.

Every year, the poultry industry experiences significant economic losses due to epidemics of Newcastle disease virus (NDV). Developing new vaccines by identifying and using the immunogenic hemagglutinin-neuraminidase (HN) protein can protect the poultry industry. In the present study, the full-length HN protein was expressed in Escherichia coli (E. coli) BL21 (DE3) cells, purified via affinity chromatography and detected via western blot analysis using His-specific antibodies. The purified HN protein was further evaluated in chickens to study the immune response against NDV. The successful production of HN-specific IgY proved the activity of the purified HN protein. IgY was present in the serum of immunized chickens. However, the immune response was higher in chickens immunized with purified HN protein along with complete and incomplete adjuvants than in chickens immunized with only the HN protein. Keywords: protein; Newcastle disease virus; poultry; infectious diseases; vaccines.

Recently, genotype VII of Newcastle disease virus (NDV) has become the most prevalent NDV genotype in Asia. Here the hemagglutinin-neuraminidase (HN) gene of genotype VII NDV strains isolated in Japan was analyzed. Notably, point amino acid substitutions in the HN protein at position 347, which is located on the major linear epitope of the HN protein, were found in two strains. However, by a hemagglutination inhibition assay, major antigenic differences did not exist between the studied strains. Additionally, chickens vaccinated with the B1 strain did not exhibit clinical effects after challenge with variants possessing the substitution at position 347 (E to K), whereas all unvaccinated chickens subjected to this challenge died within 5 days.

Influenza A viruses (IAVs) occasionally cross the species barrier and adapt to novel host species. This requires readjustment of the functional balance of the sialic acid receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA) to the sialoglycan-receptor repertoire of the new host. Novel techniques have revealed mechanistic details of this HA–NA–receptor balance, emphasizing a previously underappreciated crucial role for NA in driving the motility of receptor-associated IAV particles. Motility enables virion penetration of the sialylated mucus layer as well as attachment to, and uptake into, underlying epithelial cells. As IAVs are essentially irreversibly bound in the absence of NA activity, the fine-tuning of the HA–NA–receptor balance rather than the binding avidity of IAV particles per se is an important factor in determining host species tropism., Highlights Functional properties of the IAV HA and NA proteins need to be balanced to allow penetration of the heavily sialylated mucus layer, attachment to and endocytic uptake into underlying epithelial cells, and efficient spread of progeny virions. The HA–NA–receptor balance is readjusted upon cross-species transmission as the sialoglycan repertoire differs between species. This adjustment is achieved via mutations in the receptor-binding site of HA, but may also be achieved by adaptation of the receptor-binding and -sialidase activity properties of NA. IAVs are continuously in a receptor-bound state, which is highly dynamic resulting from multivalent, low-affinity HA–SIA interactions combined with NA activity and which allows virion movement on a receptor-containing surface. Novel technological developments allow detailed kinetic analysis of the HA–NA–receptor balance.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is a highly contagious disease in chickens and is a great threat to the poultry industry. Fusion of the viral and target cell membranes is a prerequisite for NDV's entry into host cells. This process is directly mediated by the fusion (F) protein. Although several domains of F are known to regulate membrane fusion activity, the roles of the DI-DII linker (residues 376-381) of the NDV F protein in membrane fusion still remain unclear. To investigate the roles of this linker in NDV F-induced cell-cell fusion, mutations were engineered into this linker by site-directed mutagenesis. These mutants were analysed with respect to cell surface expression and membrane fusion activity. Each of the mutated F proteins in this linker was expressed at the cell surface at a similar level to wild-type (WT) F. However, most of them resulted in significant alterations in fusion activity. In particular, the mutants G377S, A378D, L379A and T380P were able to independently mediate cell fusion in the absence of HN protein in BHK-21 cells. Taken together, the results indicated that the DI-DII linker region has an important effect on the fusion activity of NDV F and mutants in this region could alter the requirement for HN for the promotion of membrane fusion.

Autophagy triggered by glycoprotein-mediated membrane fusion has been reported for several paramyxoviruses. However, the function of HN and F glycoproteins of NDV and their role in autophagy induction have not been studied. Here, we found that co-transfection of HN and F of virulent NDV rapidly induced syncytium formation and triggered a steady state autophagy flux in adenocarcinomic human alveolar basal epithelial (A549) cells and chicken embryo fibroblast (DF-1) cells. Furthermore, we clearly identified that F and HN synergistically induced autophagosome fusion with lysosomes for subsequent degradation. The seven cleavage site mutations of F significantly decreased the autophagy induction, compared with those of wildtype virulent F. RNAi and pharmacological experiments suggested that autophagy benefitted membrane fusion and syncytium formation induced by F and HN of NDV. Activated F1 co-operated with HN to stimulate AMPK kinase and downstream ULK1 activation to suppress mTORC1 signaling. Our data described the synergistic role of HN and F in the induction of completed autophagic flux through the activation of AMPK- mTORC1- ULK1 pathway.

Background Newcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs). Methods An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes. Results Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. Conclusions These results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.

Newcastle disease (ND) is considered as one of the most devastating infectious diseases targeting domestic birds and has considerable threat to the commercial poultry production. Two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), act as antigens in the virus structure and also play important roles in infecting host cells. In the current study, the expression of the chimeric HN-F protein in canola seeds and its immunogenicity in chickens were investigated. The HN-F gene was cloned downstream of the fatty acid elongase 1 (FAE1) promoter in the binary expression vector, pBI1400-HN-F, and introduced into rapeseed (Brassica napus L.) using Agrobacterium-mediated transformation. The amount of the HN-F glycoprotein was estimated up to 0.18% and 0.11% of the total soluble protein (TSP) in transgenic seeds and leaves of canola, respectively. Confirmatory analyses of 36 transgenic lines revealed that the HN-F gene was integrated into the genome. Subsequently, HN-F protein could be expressed and accumulated in the seed tissue. Specific pathogen-free (SPF) chickens immunized orally with recombinant HN-F showed a significant rise in specific and hemagglutination inhibition (HI) antibodies 35 to 42 days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.

An expanded hydrophobic cavity within the structurally constrained receptor-binding site of the Cedar virus attachment glycoprotein facilitates idiosyncratic utilization of ephrin-B1., The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)—a related henipavirus that is ostensibly nonpathogenic—possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.

Although neuraminidase (NA) is the second major viral glycoprotein of influenza virus, its immune mechanism as a vaccine target has been less considered. Here we compared the properties of antibodies and the efficacy of cross protection by N1 and N2 NA proteins, inactivated split influenza vaccines (split), and tandem repeat extracellular domain M2 on virus-like particles (M2e5x VLP). Anti-NA immune sera could confer better cross-protection against multiple heterologous influenza viruses correlating with NA inhibition activity compared to split vaccine immune sera. Whereas split vaccine was superior to NA in conferring homologous protection. NA and M2e immune sera each showed comparable survival protection. Protective efficacy by NA immune sera was lower in Fc receptor common γ-chain deficient mice but comparable in C3 complement deficient mice compared to that in wild type mice, suggesting a role of Fc receptor in NA immunity.

The antiviral mechanism of sulfated polysaccharides is supposed to prevent virus entry, which is mediated by the interactions of anionic charges on sulfated polysaccharides with positively charged domains of viral envelope glycoproteins, leading to shielding of the functional domain involved in virus attachment to cell surface receptors. But, few direct evidences were reported. In the previous study, we found that sulfated Chuanmingshen violaceum polysaccharides (sCVPS) possessed remarkable inhibitory effect against Newcastle disease virus (NDV) through inhibition of NDV attachment to host cells. Whether sCVPS bound to hemagglutinin-neuraminidase (HN) leading to inhibition of NDV attachment needs to be further clarified. The present study conducted site-directed mutagenesis of possible positively charged residues of HN, and found that mutants R197G, H199G, R363G, and R523G could significantly decrease the inhibitory effects of sCVPS on receptor binding ability through hemadsorption assay, especially R363G which suggested that binding to R363 is more effective to shield the sialic acid binding sites. Dual mutants (R363G/R197G, R363G/H199G and R363G/R523G) induced more decreased inhibitory effect of sCVPS than single mutants. The immunofluorescence study using FITC-labeled sCVPS found that the fluorescence intensity of mutants R363G and R363G/H199G were significantly decreased. The binding kinetics of sCVPS to HN measured by surface plasmon resonance indicated that sCVPS had a higher binding affinity for wild-type HN than mutants R363G and R363G/H199G. Plaque reduction study was performed using recombinant NDV with mutant HNR363G and HNR363G/H199G, which showed significantly decreased inhibitory effects of sCVPS against mutant NDV adsorption to BHK-21 cells. These results suggested that the residues R197, H199, R363, and R523 were the binding sites for sCVPS, especially R363 act as the main interaction site. The present study provided direct evidence for the theory that antiviral mechanism of sulfated polysaccharides attributed to anionic groups binding to the positively charged residues of viral proteins which led to the shielding of receptor binding sites.

Porcine rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km=0.029μM, Vmax=522.8nmolmin-1mg-1). When 3SL was used as substrate, typical cooperative kinetics were found (S50=0.15μM, Vmax=154.3nmolmin-1mg-1). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC50 of 0.24μM. PorPV neuraminidase was activated by Ca2+ and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors.

Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein., Author summary Influenza A viruses infect birds and mammals. They contain receptor-binding (HA) and receptor-destroying (NA) proteins, which are crucial determinants of host tropism and pathogenesis. It is generally accepted that the functional properties of HA and NA need to be well balanced to enable virion penetration of the receptor-rich mucus layer, binding to host cells, and release of newly assembled particles. This HA-NA-receptor balance is, however, poorly characterized resulting in part from a lack of suitable assays to measure this balance. In addition, NA is much less studied than HA. NA contains, besides its receptor-cleavage site, a 2nd receptor-binding site, which is functional in avian, but not in human viruses. We now show that this 2nd receptor-binding site prefers binding to avian-type receptors and promotes cleavage of substrates carrying this receptor. Furthermore, by using novel assays, we established an important role for this site in the HA-NA-receptor balance of virus particles as it contributes to receptor binding and cleavage by virions, the latter of which is required for virion movement and self-elution from receptors. The results may provide an explanation for the rapid loss of a functional 2nd receptor-binding site in human pandemic viruses.

International audience; AbstractThe fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion–fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.

A codon modification strategy was used to attenuate the avian pathogenicity of an oncolytic mesogenic Newcastle disease virus (NDV) by targeting the three major virulence factors: the fusion (F) protein, hemagglutinin neuraminidase (HN) and phosphoprotein (P). Recoding the F and HN genes with rare codons greatly reduced expression of both F and HN proteins and resulted in their low incorporation into virions. The F and HN recoded virus was partially attenuated in chickens even when the F protein cleavage site was modified. Full attenuation was achieved when the 5' portion of the P gene was recoded. The recoded P, F and HN triple gene mutant exhibited delayed cell death in human cancer cells with prolonged expression of a GFP transgene. While this engineered attenuated NDV strain has lower oncolytic potency, its capacity for prolonged transgene expression may allow its use as a vaccine or gene delivery vector.

Receptor destruction has been considered one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach to or penetrate the infected cells and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that the α2,3-linked sialic acids were specifically reduced in the SeV-infected cells, but the level of α2,6-linked sialic acids had not changed. As infection with IAV removed both α2,3- and α2,6-linked sialic acids, especially α2,3-linked sialic acids, IAV-infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, α2,3-linked sialic acids, is relevant to homologous interference by SeV. IMPORTANCE Viral interference is a classically observed phenomenon, but the precise mechanism is not clear. Using SeV interference, we provide concrete evidence that reduction of the α2,3-linked sialic acid receptor by the HN of SeV is closely related with viral interference. Since SeV infection resulted in decrease of only α2,3-linked sialic acids, IAV, which also utilized α2,6-linked sialic acids to initiate infection, superinfected the SeV-infected cells. In contrast, SeV could not superinfect the IAV-infected cells because both α2,3- and α2,6-linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, many researchers have reported that lentogenic NDV is a safe and effective agent for human cancer therapy. It had been demonstrated that the amino acid sequence of the fusion protein cleavage site is a major factor in the pathogenicity and anti-tumor efficacy of rNDV. However, the role of Hemagglutinin-Neuraminidase (HN) gene that contributes to virulence and anti-tumor efficacy remains undefined. To assess the role of HN gene in virus pathogenicity and anti-tumor efficacy, a reverse genetic system was developed using the lentogenic NDV Clone30 strain to provide backbone for gene exchange. Chimeric virus (rClone30-Anh(HN)) created by exchange of the HN gene of lentogenic strain Clone30 with HN gene of mesogenic strain produce no significant changes in virus pathogenicity as assessed by conducting the mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. In vitro, infection with chimeras could induce the formation of syncytium relative significantly in HepG2 cells. Furthermore, chimeras was shown to induce the cell apoptosis via MTT and Annexin V-PI assays, reduce mitochondrial membrane potential and increase the mRNA transcription level of caspase 3. In vivo, ICR mice carrying tumor of hepatoma H22 cells were treated via intratumoral injection of chimeric virus. The treatment of chimera shows an obvious suppression in tumor volume. These results suggest that it could be an ideal approach to enhance the antitumor ability of Newcastle disease virus and highlighted the potential therapeutic application of rClone30-Anh(HN) as a viral vector to deliver foreign genes for treatment of cancers.

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.

Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV.

Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.

Although a polybasic HA 0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA 0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA 0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA 0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.

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.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011–2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011–2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound,in silicoanalyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314–361 and 474–490) and fusion protein (region 440–484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314–316 or in the region 474–490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests that humans carrying neutralizing antibodies against MuV are protected from infection by batMuV. The close functional relationship between MuV and batMuV is demonstrated by cooperation of the respective HN and F proteins to induce syncytium formation in heterologous expression studies. An interesting feature of the glycoproteins of batMuV is the downregulation of the fusion activity by the signal peptide of F, which has not been reported for other paramyxoviruses. These results are important contributions for risk assessment and for a better understanding of the replication strategy of batMuV.

Galectin-1 is an important immunoregulatory factor and can mediate the host–pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro. We confirmed that CG-1B interacted with NDV hemagglutinin–neuraminidase (HN) glycoprotein, in which the specific G4 N-glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N-glycans on HN glycoprotein.

The fusion (F) protein of Newcastle disease virus (NDV) affects viral infection and pathogenicity through mediating membrane fusion. Previously, we found NDV with increased fusogenic activity in which contained T458D or G459D mutation in the F protein. Here, we investigated the effects of these two mutations on viral infection, fusogenicity and pathogenicity. Syncytium formation assays indicated that T458D or G459D increased the F protein cleavage activity and enhanced cell fusion with or without the presence of HN protein. The T458D- or G459D-mutated NDV resulted in a decrease in virus replication or release from cells. The animal study showed that the pathogenicity of the mutated NDVs was attenuated in chickens. These results indicate that these two single mutations in F altered or diminished the requirement of HN for promoting membrane fusion. The increased fusogenic activity may disrupt the cellular machinery and consequently decrease the virus replication and pathogenicity in chickens.

Two transmembrane glycoproteins form spikes on the surface of Sendai virus, a member of the Respirovirus genus of the Paramyxovirinae subfamily of the Paramyxoviridae family: the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. HN, in contrast to F, is dispensable for viral particle production, as normal amounts of particles can be produced with highly reduced levels of HN. This HN reduction can result from mutation of an SYWST motif in its cytoplasmic tail to AFYKD. HN AFYKD accumulates at the infected cell surface but does not get incorporated into particles. In this work, we derived experimental tools to rescue HN AFYKD incorporation. We found that coexpression of a truncated HN harboring the wild-type cytoplasmic tail, the transmembrane domain, and at most 80 amino acids of the ectodomain was sufficient to complement defective HN AFYKD incorporation into particles. This relied on formation of disulfide-bound heterodimers carried out by the two cysteines present in the HN 80-amino-acid (aa) ectodomain. Finally, the replacement of the measles virus H cytoplasmic and transmembrane domains with the corresponding HN domains promoted measles virus H incorporation in Sendai virus particles.

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.

Influenza A viruses possess two surface glycoproteins, hemagglutinin (HA), which binds to sialic-acid-containing receptors, and neuraminidase (NA), which removes sialic acid from host cells. It is well established that the HA-NA functional balance regulates the efficiency of virus replication. Here, we selected a plaque variant of the WSN (H1N1) strain that grew better than the wild-type virus in NA-expressing MDCK cell culture. A reverse genetics study revealed that the single mutation HA E190K, which occurs infrequently in naturally isolated H1N1 viruses, was responsible for the phenotype of this variant. Receptor assays indicated that this mutation did not affect the receptor specificity of HA but enhanced its receptor-binding affinity, resulting in altered HA-NA functional balance relative to that of the wild-type virus. We also found that this variant replicated in nasal turbinates at an equivalent level but in lungs at a lower level compared with wild-type virus, demonstrating its attenuation in mice. Together, our data demonstrated the importance of the HA-NA functional balance for influenza virus replication in an in vivo biological setting.

A virulent Newcastle disease virus strain was isolated from diseased chickens in Shanghai, China. The isolated strain was initially characterized as highly virulent because of a short mean death time in embryonated chicken eggs and specific-pathogen-free chickens and was typed as neurotropic by intracloacal inoculation of chickens. The isolated strain had a dibasic amino acid motif in the fusion protein cleavage site sequence required for systemic replication in the host cell. The strain fell into subgenotype VIId by phylogenetic analysis of the fusion protein gene. Although these results demonstrated some sequence similarity between the isolated strain and strains responsible for outbreaks of Newcastle disease in China and Taiwan, the unusually high mortality (86.4%) set this strain aside from other VII strains. Finally, a cross-protection assay demonstrated that La Sota and clone 30 live vaccines could not protect chickens from infection with the isolated strain, with a zero survival rate being observed when chickens were challenged with a high dose of virulent VIId virus.

The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) constitutes, together with the fusion glycoprotein, the main surface antigen of this avian pathogen, which causes a highly contagious disease, relevant economically worldwide. The purpose of this work was to obtain the HN glycoprotein as a soluble antigen in culture supernatants of recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells and to evaluate its application to the development of a recombinant enzyme-linked immunosorbent assay (rELISA) for the analysis of chicken sera. A transfer vector for baculovirus containing the sequence of a melittin signal peptide was constructed and the sequence coding for HN protein without its own signal peptide was cloned. The recombinant protein was secreted and recovered easily from the culture medium of Sf9-infected cells. The recombinant protein was evaluated as antigen for ELISA coating the plates with the recovered HN using 79 positive and 142 negative samples. The Cohen kappa value resulted 0.91, indicating excellent agreement between the rELISA and the hemagglutinin inhibition tests. The rELISA was also compared with a commercial ELISA, finding high levels of agreement between both assays. The present results show that the cloning strategy developed yielded the HN protein free in the cell culture supernatant and that the recombinant protein retained its reactivity with anti-NDV HN antibodies in chicken sera.

Plasmid-encoded DNA vaccine is a novel and potentially powerful tool for cancer therapy. Since the strength of immune responses induced by DNA vaccine is usually rather low, a major goal in DNA vaccine development is to enhance vaccine-induced immunity. In this study, we investigated an approach based on the use of a viral surface protein with pleiotropic function as a potential immune enhancer. To this end, we prepared bicistronic DNA plasmids encoding the hemagglutinin–neuraminidase (HN) protein of Newcastle Disease Virus in addition to a tumor target antigen. We demonstrate a higher tumor antigen-specific T cell-mediated immune response and a lower humoral response upon vaccination with a bicistronic DNA plasmid with incorporated HN gene. In a prophylactic immunization tumor model with the surrogate tumor antigen beta-galactosidase (β-gal) and in a therapeutic immunization tumor model with the xenogeneic tumor antigen human Epithelial Cell Adhesion Molecule (hEpCAM), HN gene incorporation into the DNA vaccine led to better survival and tumor regression in mice. There was also cross protection in the therapeutic tumor model against a second challenge by the parental mouse mammary carcinoma cells in mice vaccinated with the bicistronic plasmids. This is the first report describing the HN protein as an immunomodulator for enhanced antigen-specific T cell responses via DNA plasmids. The results show that co-expression of HN with a tumor target antigen through bicistronic vectors ensures precise temporal and spatial co-delivery to direct anti-tumor immune responses preferentially towards Th1.

The aim of this study was to evaluate the disease-inducing ability of four chimeric Newcastle disease viruses (NDV) by clinicopathological assessment. The infectious clones were previously generated by insertion of hemagglutinin-neuraminidase (HN) and/or fusion (F) genes from virulent strains (Turkey North Dakota and California 02) into a mesogenic strain (Anhinga) backbone. Groups of 4-week-old chickens were inoculated via eye drop instillation, clinical signs were monitored daily, and necropsies with collection of tissues were performed at 2, 5, 10, and 14 days post infection. Tissue sections were evaluated for histopathology and immunohistochemistry for NDV nucleoprotein. All viruses replicated successfully in the natural host, although viral recovery, seroconversion, and extent of immunohistochemical staining were greatest from birds infected with those viruses containing both F and HN genes from the same virulent virus. There was minimal to no increase in clinicopathologic disease due to infection with the chimeras compared to the recombinant backbone. However, all birds developed histological evidence of encephalitis. The results suggest that the inherent virulence of Turkey North Dakota and California 2002 strains is due to more than the simple presence of their F and HN genes.

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.

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.

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.

For paramyxoviruses, entry requires a receptor-binding protein (hemagglutinin-neuraminidase [HN], H, or G) and a fusion protein (F). Like other class I viral fusion proteins, F is expressed as a prefusion metastable protein that undergoes a refolding event to induce fusion. HN binding to its receptor triggers F refolding by an unknown mechanism. HN may serve as a clamp that stabilizes F in its prefusion state until HN binds the target cell (the “clamp model”). Alternatively, HN itself may undergo a conformational change after receptor binding that destabilizes F and causes F to trigger (the “provocateur model”). To examine F-HN interactions by bimolecular fluorescence complementation (BiFC), the cytoplasmic tails of parainfluenza virus 5 (PIV5) F and HN were fused to complementary fragments of yellow fluorescent protein (YFP). Coexpression of the BiFC constructs resulted in fluorescence; however, coexpression with unrelated BiFC constructs also produced fluorescence. The affinity of the two halves of YFP presumably superseded the F-HN interaction. Unexpectedly, coexpression of the BiFC F and HN constructs greatly enhanced fusion in multiple cell types. We hypothesize that the increase in fusion occurs because the BiFC tags bring F and HN together more frequently than occurs in a wild-type (wt) scenario. This implies that normally much of wt F is not associated with wt HN, in conflict with the clamp model for activation. Correspondingly, we show that wt PIV5 fusion occurs in an HN concentration-dependent manner. Also inconsistent with the clamp model are the findings that BiFC F does not adopt a postfusion conformation when expressed in the absence of HN and that HN coexpression does not provide resistance to the heat-induced triggering of F. In support of a provocateur model of F activation, we demonstrate by analysis of the morphology of soluble F trimers that the hyperfusogenic mutation S443P has a destabilizing effect on F.

The recent global resurgence of mumps has drawn attention to the continued need for robust mumps immunization programs. Unfortunately, some vaccines derived from inadequately attenuated vaccine strains of mumps virus have caused meningitis in vaccinees, leading to withdrawal of certain vaccine strains from the market, public resistance to vaccination, or in some cases, cessation of national mumps vaccination programs. The most widely implicated mumps vaccine in cases of postvaccination meningitis is derived from the Urabe AM9 strain, which remains in use in some countries. The Urabe AM9 vaccine virus has been shown to exhibit a considerable degree of nucleotide and amino acid heterogeneity. Some studies have specifically implicated variants containing a lysine residue at amino acid position 335 in the hemagglutinin-neuraminidase (HN) protein with neurotoxicity, whereas a glutamic acid residue at this position was associated with attenuation. To test this hypothesis we generated two modified Urabe AM9 cDNA clones coding either for a lysine or a glutamic acid at position 335 in the HN gene. The two viruses were rescued by reverse genetics and characterized in vitro and in vivo. Both viruses exhibited similar growth kinetics in neuronal and non-neuronal cell lines and were of similar neurotoxicity when tested in rats, suggesting that amino acid 335 is not a crucial determinant of Urabe AM9 growth or neurovirulence.

Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects young children, but no vaccines or antiviral drugs against it have yet been developed. We developed a mouse model to evaluate the efficacies of the novel parainfluenza virus hemagglutinin-neuraminidase (HN) inhibitors BCX 2798 and BCX 2855 against a recombinant Sendai virus (rSeV) in which the fusion (F) and HN surface glycoproteins (FHN) were replaced by those of hPIV-3 [rSeV(hPIV-3FHN)]. In the prophylaxis model, 129X1/SvJ mice were infected with a 90% or 20% lethal dose of the virus and were treated intranasally for 5 days with 10 mg/kg of body weight/day of either compound starting 4 h before infection. Prophylactic treatment of the mice with either compound did not prevent their death in a 90% lethality model of rSeV(hPIV-3FHN) infection. However, it significantly reduced the lung virus titers, the amount of weight lost, and the rate of mortality in mice infected with a 20% lethal virus dose. In the therapy model, mice were infected with a nonlethal dose of the virus (100 PFU/mouse) and were treated intranasally with 1 or 10 mg/kg/day of either compound for 5 days starting at 24 or 48 h postinfection. Treatment of the mice with either compound significantly reduced the virus titer in the lungs, subsequently causing a reduction in the number of immune cells and the levels of cytokines in the bronchoalveolar lavage fluid and histopathologic changes in the airways. Our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of hPIV-3 HN in our mouse model and may be promising candidates for the prophylaxis and treatment of hPIV-3 infection in humans.

Virulent Newcastle disease virus isolates from the 1971 and 2002 U.S. outbreaks are of the same serotype but a different genotype than current vaccine strains. Prior experiments with inactivated vaccines in chickens show significantly less virus shed in birds vaccinated with a homologous vaccine (same genotype as challenge) compared to chickens vaccinated with genotypically heterologous vaccines. Subsequent experiments have compared the protection induced in chickens by live vaccines of B1 and LaSota (genotype II), Ulster (genotype I), and recombinant viruses that express the hemagglutinin neuraminidase gene (HN) or the HN and fusion gene (F) of CA 2002 (genotype V). Vaccinates were challenged with virulent viruses CA 2002 (genotype V) or Texas GB (TXGB, genotype II). After challenge with CA 2002 the birds vaccinated with a live recombinant genotype V virus containing the HN of CA 2002 shed significantly less virus in oropharyngeal swabs compared to B1 and had fewer birds shedding virus compared to B1, LaSota, and Ulster vaccinates. After challenge with CA 2002 birds vaccinated with the recombinant containing both the HN and F of CA 2002 (rA-CAFHN) shed less virus, and fewer birds shed virus compared to LaSota-vaccinated birds. TXGB-challenged LaSota-vaccinated birds shed less virus, and fewer birds shed virus compared to TXGB-challenged rA-CAFHN-vaccinated birds. Genotypic differences between vaccine and challenge did not diminish ability of vaccines to protect against disease, but genotypic similarity did reduce virus shed and may reduce transmission. The development and use of vaccines of the same genotype as the expected field challenge may provide an additional tool for control of this important poultry pathogen.

Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV.

“Blue eye disease” is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signs and corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening and adult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene of several porcine rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did not produce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6–PAC9 viruses, whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed that the main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3 viruses, as well as A511K and E514K presented in PAC6–PAC9 viruses considerably modified the properties of the HN protein surface. The increased positive charge of the HN protein of PAC6–PAC9 viruses seems to be associated with their increased neurovirulence.

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the Fusion protein. The interrelationship between the receptor binding and fusion-triggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site's receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A mutation coding for the amino acid change E335 to K is frequently found in the hemagglutinin-neuraminidase (HN) gene of Urabe AM9 mumps viruses isolated during post-vaccination meningitis cases. To identify if this mutation modifies the biological activities of the HN glycoprotein, two variants of Urabe AM9 vaccine differing at amino acid 335 (HN-E335 and HN-K335) were isolated and their receptor-binding specificity was determined by means of competence assays. Pre-incubation of the viruses with sialic acids inhibited both syncytia formation in Vero cells and replication in SH-SY5Y cells. Thus, HN-K335 showed higher affinity towards sialylalpha2,6lactose, whereas HN-G335 preferred sialylalpha2,3lactose. These results are relevant because a high expression of sialylalpha2,6lactose in nerve cells was confirmed by means of Sambucus nigra lectin-cytochemistry. In addition, kinetics assays showed that HN-K335 and HN-E335 also differ in their hydrolysis rate (Vmax values of 37.5 vs. 3.5 nmol min-1mg-1, respectively). Therefore, HN-K335 variant presented a neuraminidase activity level 11-fold higher than that of HN-E335 variant. In conclusion, the mutation affects the receptor-binding and neuraminidase activities of Urabe AM9 mumps virus variants.

Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut(+) phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67 nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test.