Hemagglutinin-neuraminidase Research Articles

The Epidemiological Characteristics of the Korean Bat Paramyxovirus between 2016 and 2019.

Bats are considered reservoirs of severe emerging human pathogens. Notably, bats host major mammalian paramyxoviruses from the family Paramyxoviridae, order Mononegavirales. In this study, paramyxoviruses were investigated by reverse transcription semi-nested polymerase chain reaction (RT-semi-nested PCR) and reverse transcription polymerase chain reaction (RT-PCR), based on the RT-semi-nested PCR using the consensus paramyxovirus primers targeting the RNA dependent-RNA-polymerase (RdRp) region. In addition, RT-PCR was performed using newly designed primers targeting regions of the fusion protein (F) and hemagglutinin-neuraminidase (HN). The dominant bat species in the collection site of paramyxoviruses were Miniopterus schreibersii, Myotis macrodactylus, Myotis petax, and Rhinolophus ferrumequinum. Paramyxoviruses were detected in four samples in 2016 and six in 2019. Meanwhile, in samples collected in 2017 and 2018, no paramyxoviruses were detected. Phylogenetic analysis based on the partial nucleotide sequences of RdRp, F, and HN proteins suggested that the viruses belonged to the proposed genus Shaanvirus. In conclusion, this study revealed that bat paramyxoviruses in Korea belonged to a single genus and circulated sporadically in several provinces, including Chungbuk, Gangwon, Jeju, and Jeonnam.

Development of PLGA-PEG-PLGA Hydrogel Delivery System for Enhanced Immunoreaction and Efficacy of Newcastle Disease Virus DNA Vaccine.

The highly contagious Newcastle disease virus (NDV) continues to threaten poultry all over the world. The NDV DNA vaccine is a promising solution to the current Newcastle disease (ND) challenges, and thus an efficient delivery system should be developed to facilitate the efficacy of DNA vaccines. In this study, we developed a DNA vaccine delivery system consisting of a triblock copolymer of poly(lactide co-glycolide acid) and polyethylene glycol (PLGA-PEG-PLGA) hydrogel in which the recombinant NDV hemagglutinin-neuraminidase (HN) plasmid was encapsulated. Its characteristics, security, immune responses, and efficacy against highly virulent NDV were detected. The results showed that the plasmids were gradually released in a sustained manner from the hydrogel, which improved the biological stability of the plasmids and demonstrated a high biocompatibility. The plasmids, when they were incorporated into the hydrogel delivery system, enhanced immune activation and provided 100% protection against the highly virulent NDV strain. Furthermore, we proved that this NDV DNA hydrogel vaccine could improve the lymphocyte proliferation and increase the immunological cytokine production via the PI3K/Akt pathway. These results indicate that the PLGA-PEG-PLGA thermosensitive hydrogel could be a promising delivery system for the NDV DNA vaccine in order to achieve a sustained supply of plasmids and induce potent immune responses.

Active virological surveillance in backyard ducks in Bangladesh: detection of avian influenza and gammacoronaviruses

ABSTRACT Domestic waterfowl play an important role in the perpetuation and transmission of avian pathogens including avian influenza viruses (AIV) of low and high pathogenicity, which pose severe economic and public health concerns in Bangladesh. This study focused on active surveillance of several avian viral pathogens with a special reference to AIV in selected backyard duck populations in Bangladesh. A total of 500 pooled oropharyngeal and cloacal samples from individual ducks of four districts were tested by real time PCRs for the presence of AIV, avian avulavirus-1, anatid herpesvirus-1, avian parvovirus, avian bornavirus and avian coronavirus. The investigation identified 27 (5.4%) ducks positive for AIV and 12 (2.4%) positive for avian coronavirus. In 13 samples, RNA specific for AIV H4N6 was detected. Phylogenetic analysis of the AIV haemagglutinin H4 and neuraminidase N6 genes suggested a clustering of Bangladeshi AIV H4N6 in Eurasian lineage group 2. Other AIV positive samples had very low virus loads (Cq > 36) and were not subtyped. Coronaviral sequences of a fragment of the polymerase gene were related to Eurasian-Australian duck gamma-coronaviruses. Our current active surveillance in free-range domestic backyard ducks in Bangladesh failed to detect highly pathogenic (HP) AIV in contrast to our previous passive monitoring study. Nevertheless, active monitoring of domestic duck populations may be important to highlight presence and transmission dynamics of economically less important AIV that still may serve as reassortment partners for the generation of new HP and zoonotic AIV. RESEARCH HIGHLIGHTS Active surveillance for viral pathogens in domestic free-range backyard ducks. Detection of avian influenza virus subtype H4N6. First identification of avian gammacoronavirus in ducks in Bangladesh.

Screening and mechanistic study of key sites of the hemagglutinin-neuraminidase protein related to the virulence of Newcastle disease virus

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.

Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy

: Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells. A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole. Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein. Results showed efficient production of the bsAb in E. coli for future large-scale purification.

Chimeric Newcastle Disease Virus-like Particles Containing DC-Binding Peptide-Fused Haemagglutinin Protect Chickens from Virulent Newcastle Disease Virus and H9N2 Avian Influenza Virus Challenge.

Newcastle disease virus (NDV) and H9N2 subtype Avian influenza virus (AIV) are two notorious avian respiratory pathogens that cause great losses in the poultry industry. Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses, while an attenuated live vaccine bears the risk of mutation. Dendritic cell (DC) targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens. In this study, DC-binding peptide (DCpep)-decorated chimeric virus-like particles (cVLPs), containing NDV haemagglutinin-neuraminidase (HN) and AIV haemagglutinin (HA), were developed as a DC-targeting mucosal vaccine candidate. DCpep-decorated cVLPs activated DCs in vitro, and induced potent immune stimulation in chickens, with enhanced secretory immunoglobulin A (sIgA) secretion and splenic T cell differentiation. 40μg cVLPs can provide full protection against the challenge with homologous, heterologous NDV strains, and AIV H9N2. In addition, DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly, as indicated by robust sIgA secretion and a reduced virus shedding period. Taken together, this chimeric VLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.

The Immunogenicity of a Novel Chimeric Hemagglutinin-Neuraminidase-Fusion Antigen from Newcastle Disease Virus by Oral Delivery of Transgenic Canola Seeds to Chickens.

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 42days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.

Study on the Antiviral Activities and Hemagglutinin-Based Molecular Mechanism of Novel Chlorogenin 3-O-β-Chacotrioside Derivatives Against H5N1 Subtype Viruses.

The objective of this study was to investigate the inhibitory effect of chlorogenin 3-O-β-chacotrioside derivatives against H5N1 subtype of the highly pathogenic avian influenza (HPAI) viruses and its molecular mechanism. A series of novel small molecule pentacyclic triterpene derivatives were designed and synthesized and their antiviral activities on HPAI H5N1 viruses were detected. The results displayed that the derivatives UA-Nu-ph-5, XC-27-1 and XC-27-2 strongly inhibited wild-type A/Duck/Guangdong/212/2004 H5N1 viruses with the IC50 values of 15.59 ± 2.4 μM, 16.83 ± 1.45 μM, and 12.45 ± 2.27 μM, respectively, and had the selectivity index (SI) > 3, which was consistent with the efficacy against A/Thailand/kan353/2004 pseudo-typed viruses. Four dealt patterns were compared via PRNT. The prevention dealt pattern showed the strongest inhibitory effects than other patterns, suggesting that these derivatives act on the entry process at the early stages of H5N1 viral infection, providing protection for cells against infection. Further studies through hemagglutinin inhibition (HI) and neuraminidase inhibitory (NAI) assay confirmed that these derivatives inhibited H5N1 virus replication by interfering with the viral hemagglutinin function. The derivatives could recognize specifically HA protein with binding affinity constant KD values of 2.57 × 10−4 M and 3.67 × 10−4 M. In addition, through site-directed mutagenesis combined with a pseudovirion system, we identified that the high-affinity docking sites underlying interaction were closely associated with amino acid residues I391 and T395 of HA. However, the potential binding sites of the derivatives with HA did not locate at HA1 sialic acids receptor binding domain (RBD). Taken together, these study data manifested that chlorogenin 3-O-β-chacotrioside derivatives generated antiviral effect against HPAI H5N1 viruses by targeting the hemagglutinin fusion machinery.

Genetic diversity and evolutionary analysis of human respirovirus type 3 strains isolated in Kenya using complete hemagglutinin-neuraminidase (HN) gene.

Human respirovirus type 3 (HRV3) is a leading etiology of lower respiratory tract infections in young children and ranks only second to the human respiratory syncytial virus (HRSV). Despite the public health importance of HRV3, there is limited information about the genetic characteristics and diversity of these viruses in Kenya. To begin to address this gap, we analyzed 35 complete hemagglutinin-neuraminidase (HN) sequences of HRV3 strains isolated in Kenya between 2010 and 2013. Viral RNA was extracted from the isolates, and the entire HN gene amplified by RT-PCR followed by nucleotide sequencing. Phylogenetic analyses of the sequences revealed that all the Kenyan isolates grouped into genetic Cluster C; sub-clusters C1a, C2, and C3a. The majority (54%) of isolates belonged to sub-cluster C3a, followed by C2 (43%) and C1a (2.9%). Sequence analysis revealed high identities between the Kenyan isolates and the HRV3 prototype strain both at the amino acid (96.5–97.9%) and nucleotide (94.3–95.6%) levels. No amino acid variations affecting the catalytic/active sites of the HN glycoprotein were observed among the Kenyan isolates. Selection pressure analyses showed that the HN glycoprotein was evolving under positive selection. Evolutionary analyses revealed that the mean TMRCA for the HN sequence dataset was 1942 (95% HPD: 1928–1957), while the mean evolutionary rate was 4.65x10-4 nucleotide substitutions/site/year (95% HPD: 2.99x10-4 to 6.35x10-4). Overall, our results demonstrate the co-circulation of strains of cluster C HRV3 variants in Kenya during the study period. This is the first study to describe the genetic and molecular evolutionary aspects of HRV3 in Kenya using the complete HN gene.

Hijacking the Fusion Complex of Human Parainfluenza Virus as an Antiviral Strategy.

The receptor binding protein of parainfluenza virus, hemagglutinin-neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein prior to receptor engagement of HN at the host cell membrane inactivate the virus. We previously identified small molecules that interact with HN and induce it to activate F in an untimely fashion, validating a new antiviral strategy. To obtain highly active pretriggering candidate molecules we carried out a virtual modeling screen for molecules that interact with sialic acid binding site II on HN, which we propose to be the site responsible for activating F. To directly assess the mechanism of action of one such highly effective new premature activating compound, PAC-3066, we use cryo-electron tomography on authentic intact viral particles for the first time to examine the effects of PAC-3066 treatment on the conformation of the viral F protein. We present the first direct observation of the conformational rearrangement induced in the viral F protein.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), upon binding to its cell receptor, triggers conformational changes in the fusion protein (F). This action of HN activates F to reach its fusion-competent state. Using small molecules that interact with HN, we can induce the premature activation of F and inactivate the virus. To obtain highly active pretriggering compounds, we carried out a virtual modeling screen for molecules that interact with a sialic acid binding site on HN that we propose to be the site involved in activating F. We use cryo-electron tomography of authentic intact viral particles for the first time to directly assess the mechanism of action of this treatment on the conformation of the viral F protein and present the first direct observation of the induced conformational rearrangement in the viral F protein.

Role of the head-stalk linker region (aa115-122) of Newcastle disease virus haemagglutinin-neuraminidase in regulating fusion triggering.

To gain insights into the role of the head-stalk linker region in the fusion triggering, we constructed mutants by deleting or substituting the linker region (115-NGAANNSG-122) of Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) with the corresponding sequences of other paramyxoviruses. The results showed that these HN mutants exhibited different levels of fusion-triggering activity, but most of them maintained comparable levels with wide-type HN in both receptor recognition and neuraminidase activity. We tried to figure out reasons for fusion alteration through assessing the expression and the oligomeric state of HN mutants. Moreover, four mutants with significant fusion changes were introduced into the headless HN stem (HN1-123) to intensively investigate the role of the linker region in fusion triggering. Consequently, the stability of HN oligomers and the structural integrity of the 4 helical-bundle of stalk have complicated influences on the alteration of fusion-triggering activities for different mutants. These data suggested that the head-stalk linker could regulate the fusion triggering at both full-length and headless HN levels.

E. coli expression and immunological assessment of expressed recombinant Newcastle disease virus hemagglutinin-neuraminidase protein in chickens.

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.

Neutralizing and Neuraminidase Antibodies Correlate With Protection Against Influenza During a Late Season A/H3N2 Outbreak Among Unvaccinated Military Recruits.

BackgroundAntibodies that inhibit hemagglutination have long been considered a correlate of protection against influenza, but these antibodies are only a subset of potentially protective antibodies. Neutralizing and neuraminidase antibodies may also contribute to protection, but data on their associations with protection are limited.MethodsWe measured preoutbreak hemagglutinin pseudovirus neutralization (PVN) and neuraminidase inhibition (NAI) antibody titers in unvaccinated military recruits who experienced an H3N2 influenza outbreak during training. We conducted a case-control study to investigate the association between titers and protection against influenza illness or H3N2-associated pneumonia using logistic regression.ResultsWith every 2-fold increase in PVN titer, the odds of medically attended polymerase chain reaction–confirmed H3N2 infection (H3N2+) decreased by 41% (odds ratio [OR], 0.59; 95% confidence interval [CI], .45 to .77; P < .001). Among those who were H3N2+, the odds for pneumonia decreased by 52% (OR, 0.48; CI, .25 to .91; P = .0249). With every 2-fold increase in NAI titer, the odds of medically attended H3N2 infection decreased by 32% (OR, 0.68; 95% CI, .53 to .87; P = .0028), but there was no association between NAI titers and H3N2-associated pneumonia. There was also no synergistic effect of PVN and NAI antibodies.ConclusionsPVN and NAI titers were independently associated with reduced risk of influenza illness. NAI titers associated with protection had greater breadth of reactivity to drifted strains than PVN titers. These findings show that PVN and NAI titers are valuable biomarkers for assessing the odds of influenza infection.

Roles of the highly conserved amino acids in the second receptor binding site of the Newcastle disease virus HN protein

BackgroundThe paramyxovirus haemagglutinin-neuraminidase (HN) is a multifunctional protein that is responsible for attachment to receptors, removal of receptors from infected cells to prevent viral self-aggregation (neuraminidase, NA) and fusion promotion. It is commonly accepted that there are two receptor binding sites in the globular head of HN, and the second receptor binding site is only involved in the function of receptor binding and fusion promotion.Methods10 conserved residues in the second receptor binding site of Newcastle disease virus (NDV) HN were chosen and substituted to alanine (A). The desired mutants were examined to detect the functional change in hemadsorption (HAD) ability, NA activity and fusion promotion ability.ResultsThe HAD and fusion promotion ability of mutants C172A, R174A, C196A, D198A, Y526A and E547A were abolished. Compared with wild-type (wt) HN, the HAD of mutants T167A, S202A and R516A decreased to 55.81, 44.53, 69.02%, respectively, and the fusion promotion ability of these three mutants decreased to 54.74, 49.46, 65.26%, respectively; however, mutant G171A still maintained fusion promotion ability comparable with wt HN but had impaired HAD ability. All the site-directed mutations altered the NA activity of NDV HN without affecting protein cell surface expression.ConclusionsThe data suggest that mutants C172A, R174A, C196A, D198A, Y526A and E547A do not allow the conformational change that is required for fusion promotion ability and HAD activity, while the other mutants only affect the conformational change to a limited extent, except mutant G171A with intact fusion promotion ability. Overall, the conserved amino acids in the second receptor binding site, especially residues C172, R174, C196, D198, Y526 and E547, are crucial to normal NDV HN protein function.

Molecular evolution of the hemagglutinin-neuraminidase (HN) gene in human respirovirus 3

Human respirovirus 3 (HRV3) is a major causative agent of acute respiratory infections in humans. HRV3 can manifest as a recurrent infection, although exactly how is not known. In the present study, we conducted detailed molecular evolutionary analyses of the major antigen-coding hemagglutinin-neuraminidase (HN) gene of this virus detected/isolated in various countries. We performed analyses of time-scaled evolution, similarity, selective pressure, phylodynamics, and conformational epitope prediction by mapping to HN protein models. In this way, we estimated that a common ancestor of the HN gene of HRV3 and bovine respirovirus 3 diverged around 1815 and formed many lineages in the phylogenetic tree. The evolutionary rates of the HN gene were 1.1 × 10−3 substitutions/site/year, although the majority of these substitutions were synonymous. Some positive and many negative selection sites were predicted in the HN protein. Phylodynamic fluctuations of the gene were observed, and these were different in each lineage. Furthermore, most of the predicted B cell epitopes did not correspond to the neutralization-related mouse monoclonal antibody binding sites. The lack of a link between the conformational epitopes and neutralization sites may explain the naturally occurring HRV3 reinfection.

The DI-DII linker of human parainfluenza virus type 3 fusion protein is critical for the virus.

Human parainfluenza virus type 3 (HPIV3) causes the majority of childhood viral pneumonia around the world. Fusing the viral and target cell membranes is crucial for its entry into target cells, and the fusion process requires the concerted actions of two viral glycoproteins: hemagglutinin-neuraminidase (HN) and fusion (F) protein. After binding to the cell surface receptor, sialic acids, HN triggers F to undergo large conformational rearrangements to execute the fusion process. Although it has been reported that several domains of F had important impacts on regulating the membrane fusion activity, what role the DI-DII linker (residues 369-374, namely L1 linker) of the HPIV3 F protein plays in the fusion process still remains confused. We have obtained three chimeric mutant proteins (Ch-NDV-L1, Ch-MV-L1, Ch-HPIV1-L1) containing the full length of HPIV3 F protein but their corresponding DI-DII linker derived from the F protein of Newcastle disease virus (NDV), Measles virus (MV), and Human parainfluenza virus type 1 (HPIV1), respectively. One deletion mutant protein (De-L1), whose DI-DII linker was deleted, has been established simultaneously. Then vaccinia virus-T7 RNA polymerase transient expression system and standard plasmid system were utilized to express the mutant F proteins in BHK-21 cells. These four mutants were determined for membrane fusogenic activity, cell surface expression level, and total mutant F protein expression. All of them resulted in a significant reduction in fusogenic activity in all steps of cell-cell membrane fusion process. There was no significant difference in cell surface protein expression level for the mutants compared with wild-type F. The mutant proteins with inability in fusogenic activity were all at the form of precursor protein, F0, which were not hydrolyzed by intracellular protease furin. The results above suggest that the involvement of the DI-DII linker region is necessary for the complete fusion of the membranes.

Process development for quantitation and vaccine efficacy assessment of recombinant hemagglutinin-neuraminidase

Recombinant hemagglutinin-neuraminidase (HN) based subunit vaccine, which is non-infectious and can be produced using insect cell-culture systems, is a potential alternative to conventional live and inactivated Newcastle disease virus (NDV) vaccines. However, process development for manufacture and efficacy assessment of HN subunit vaccines has been hampered by the absence of reference standards, a cornerstone for robust and sensitive quantitative analytical methods. In this work, a downstream purification strategy was developed to obtain NDV HN which was expressed with a hexa-histidine fusion tag (rHN) to facilitate detection using generic antibodies. Highly purified rHN (∼95%) attained after detergent extraction and two-stage ion-exchange-hydroxyapatite column chromatography was subsequently utilized as reference standards for quantitative ELISA development. Recovery of rHN at different stages of purification was monitored. Quantitation of rHN from crude cell lysates was performed for dose-ranging antibody response and protective efficacy studies. A higher dose (1500 ng) of rHN was correlated to a significant reduction in virus shedding and attainment of herd immunity, as indicated by a higher proportion of chickens (92%) with hemagglutination inhibition (HI) antibody titers ≥ log23. The outcome of this study, shows the importance of downstream process development in enabling robust quantitation and efficacy assessment of a recombinant subunit vaccine.

Genotype-matched Newcastle disease virus vaccine confers improved protection against genotype XII challenge: The importance of cytoplasmic tails in viral replication and vaccine design.

Although typical Newcastle disease virus (NDV) vaccines can prevent mortality, they are not effective in preventing viral shedding. To overcome this, genotype-matched vaccines have been proposed. To date, this approach has never been tested against genotype XII strains. In this study, we generated and assessed the protection against genotype XII challenge of two chimeric NDV vaccine strains (rLS1-XII-1 and rLS1-XII-2). The rLS1-XII-1 virus has the complete fusion protein (F) and the hemagglutinin-neuraminidase (HN) open reading frames replaced with those from genotype XII strain NDV/peacock/Peru/2011 (PP2011) in a recombinant LaSota (rLS1) backbone. In rLS1-XII-2 virus, cytoplasmic tails of F and HN proteins were restored to those of rLS1. In vitro evaluation showed that rLS1-XII-2 and the parental rLS1 strains replicate at higher efficiencies than rLS1-XII-1. In the first vaccine/challenge experiment, SPF chickens vaccinated with rLS1-XII-1 virus showed only 71.3% protection, whereas, rLS1 and rLS1-XII-2 vaccinated chickens were fully protected. In a second experiment, both rLS1-XII-2 and the commercial vaccine strain LaSota induced 100% protection. However, rLS1-XII-2 virus significantly reduced viral shedding, both in the number of shedding birds and in quantity of shed virus. In conclusion, we have developed a vaccine candidate capable of fully protecting chickens against genotype XII challenges. Furthermore, we have shown the importance of cytoplasmic tails in virus replication and vaccine competence.

Development of Avian Avulavirus 1 Epitope-Based Vaccine Pattern Based on Epitope Prediction and Molecular Docking Analysis: An Immunoinformatic Approach

Fusion (F) and Hemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus containing important epitopes play crucial role in stimulation of humoral and cell-mediated immunity of the host. In the present study we analyzed and compared genetic diversity of F and HN sequences related to long-term circulating sub-genotype VIIj isolates to genotype VII and II strains. In addition, F and HN epitope prediction was performed using IEDB, NetMHC 4.0 and NetMHC II 3.1 servers. Molecular docking analysis was also conducted for VIIj and LaSota isolates interacting MHC I and II molecules. Results indicated that certain specific mutations were occurred in Fusion and Hemagglutinin-neuraminidase proteins of VIIj sub-genotype. Epitope prediction study revealed that the unique variations were located on the antigenic site regions of F and HN glycoproteins. Furthermore, docking interaction analysis showed surprising results, in which defined antigenic site residues of VIIj isolates interacted to MHC molecules with higher affinity levels rather than LaSota strains used as vaccines. Our in silico results suggest that by applying certain epitope sequences inducing both immune responses, vaccination failures may be reduced and challenged vaccinated host cells may stop viral shedding beside protection from Newcastle disease infection. Thus, we recommend the introduced antigenic sites of VIIj sub-genotype in this paper would act as the great targets for epitope-based vaccine design in the region.

Molecular docking analysis of zanamavir with haem agglutinin neuraminidase of human para influenza virus type 3.

The human para influenza virus (HPIV) type 3 is an imperative respiratory virus which cause upper and lower respiratory infections. The receptor involved in the viral infection is haem agglutinin neuraminidase. It is of interest to study the interaction of haem agglutinin neuraminidase with zanamavir (4-GU-DANA), a known antiviral drug. We used the PDB structures with PDB IDs 1V2I, 1V3B, 1V3D and 1V3E for studying the interactions with zanamavir. The binding features of zanamavir with 1V2I (1.41kcal/mol) and 1V3E (11.81kcal/mol) having optimal interactions is reported for further consideration.