Hemagglutinin-neuraminidase Protein Research Articles

Monoclonal antibodies specific for the hemagglutinin-neuraminidase protein define neutralizing epitopes specific for Newcastle disease virus genotype 2.VII from Egypt

BackgroundNewcastle 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).MethodsAn 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.ResultsSix 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.ConclusionsThese 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.

The effect of the HRB linker of Newcastle disease virus fusion protein on the fusogenic activity.

Newcastle disease, designated a class A disease of poultry by the Office international des epizooties (OIE), is an acute infection caused by Newcastle disease virus (NDV). The merging of the envelope of NDV with the membrane of a target host cell is the key step in the infection pathway, which is driven by the concerted action of two glycoproteins: haemagglutinin-neuraminidase (HN) and fusion (F) protein. When the HN protein binds to the host cell surface receptor, the F protein is activated to mediate fusion. The three-dimensional structure of the F protein has been reported to have low electron density between the DIII domain and the HRB domain, and this electron-poor region is defined as the HRB linker. To clarify the contributing role of the HRB linker in the NDV F protein-mediated fusion process, 6 single amino acid mutants were obtained by site-directed mutagenesis of the HRB linker. The expression of the mutants and their abilities to mediate fusion were analysed, and the key amino acids in the HRB linker were identified as L436, E439, I450, and S453, as they can modulate the fusion ability or expression of the active form to a certain extent. The data shed light on the crucial role of the F protein HRB linker in the acquisition of a normal fusogenic phenotype.

Hemagglutinin-neuraminidase Sequence and Phylogenetic Analysis of Two Newcastle Disease Virus Isolated from Chickens in Iran.

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.

Cross-neutralization between vaccine and circulating wild-type mumps viruses in Korea

Mumps is a contagious disease caused by the mumps virus. It can be prevented using mumps vaccines, administered as a measles-mumps-rubella (MMR) vaccine. For first and second dose immunization, children aged 12–15 months and 4–6 years have been administered this vaccine since 1997 in Korea. Nevertheless, mumps outbreaks still occur in vaccinated populations worldwide. Hence, immunity against these diseases may be attenuated, or there are antigenic differences between currently available vaccine strains and circulating wild-type viruses. After the introduction of national immunization programs in Korea, mumps cases became sporadic. Viral genotypes F, H, and I have emerged since 1998 whereas the vaccine strains belong to genotype A. Here, we compared the amino acid sequences of the haemagglutinin-neuraminidase (HN) gene from wild-type viruses and the mumps vaccine and measured the cross-neutralization titers between them. We selected the F, H, and I wild-type mumps strains circulating in Korea from 1998 to 2016 and analyzed changes in the amino acid sequence of the protein encoded by the HN gene. We measured mumps virus-specific IgG and rapid focus reduction neutralization test (FRNT) titers in Korean isolates and sera obtained from 50 children aged 1–2 years who had been administered a single dose of MMR vaccine. Analysis of the HN protein sequences disclosed no changes in the glycosylation sites but did reveal 4–5 differences between the Korean isolates and the genotype A vaccine strain in terms of the neutralizing epitope sites on their HN proteins. Post-vaccination FRNT titers were significantly lower against genotypes F, H, and I than they were against genotype A. This finding highlights the possibility of a recurrence of mumps outbreaks in vaccinated populations depending on the degree of genetic conservation of the HN gene. Further research into this issue is needed to prevent the resurgence of mumps.

Role of key amino acids in the transmembrane domain of the Newcastle disease virus fusion protein.

Newcastle disease (ND), caused by the Newcastle disease virus (NDV), is transmitted by poultry with severe infectivity and a high fatality rate. The fusion (F) protein on the NDV envelope facilitates the merger of the viral and host cell membranes with the help of the homologous hemagglutinin-neuraminidase protein (HN). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion, but the key amino acids in NDV F TM domains have not been identified. Site-directed mutagenesis was utilized to change the conserved amino acids at 500, 501, 502, 505, 510, 513, 516, 519, and 520 to alanine. It was found that mutants L519 and V520 had an interrupted protein expression, decreased to below 10%, and mutants A500, I505, V513, and V516 had a hypoactive impact on fusion activity, decreased to 85.38%, 67.05%, 55.38% and 51.13% of wt F, respectively. The results indicated that the TM domain plays a vital part in the fusion activity of the NDV F protein.

Viral 2A-peptides mediate continuous transcription and self-cleavage of multiple heterologous genes in fowlpox virus vector

Applicability of viral 2A-peptides in generation of multi-cistronic transcripts to deliver separate self-cleaved proteins is well established. However, the use of viral 2A-peptides in fowlpox virus vector construction to co-express multiple heterologous genes has not been explored. To evaluate the same, a recombinant transfer plasmid pJFWPVt was constructed through two intermediate plasmid constructs, pJF7F9 and pJFHNGFP. The construction of pJF7F9 involved cloning of F7 and F9 genes of FWPV into pCI-neo with modifications in the F7-F9 intergenic region. For the construction of pJFHNGFP, a synthetic DNA adapter consisting of one synthetic early late promoter (PE/L), two viral 2A-peptides (P2A and T2A) and three multiple cloning sites (MCS1, MCS2 and MCS3) was synthesized chemically and was cloned into pUC19 to obtain pJFHNGFPi. Heterologous genes fusion (F) and haemagglutininneuraminidase (HN) of Avian Avulavirus-1 (AAv1) and marker gene AcGFP were cloned sequentially into MCS1, MCS2 and MCS3 of pJFHNGFPi to obtain pJFHNGFP. The insert (PE/L-F-P2A-HN-T2A-AcGFP) in pJFHNGFP was cloned into pJF7F9 to obtain pJFWPVt, which upon transfection in FWPV infected chicken embryo fibroblast (CEF) cells resulted in fluorescence. This confirmed the expression of AcGFP and the continuous transcription ability of viral 2A-peptides. Further, western blotting of CEF pellet showed separate protein bands of F and HN protein at 66 kDa and 74 kDa respectively, which confirmed the self-cleaving ability of viral 2A-peptides. Herein, in FWPV vector construction, continuous transcription and self-cleaving ability of viral 2A-peptides in FWPV vector construction was confirmed. This warrants scope for future viral 2A-peptide based FWPV vector construction.

Identification of a potential neutralizing linear epitope of hemagglutinin-neuraminidase in Newcastle disease virus

BackgroundThe 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.MethodsIn 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.ResultsFive 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.ConclusionThe 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.

Molecular characterisation of Newcastle disease virus exotic isolate in East Java, Indonesia

Newcastle disease virus (NDV) is ssRNA paramyxovirus causing clinical signs, varying from subclinical infections to 100% mortality in infected chickens. Haemagglutinin-neuraminidase (HN) protein has an important role related to infection and pathogenesis, therefore, the protein was characterised in this study. Samples were collected from 45 cloacal swabs of native chickens. They were isolated by inoculating in specific pathogen-free embryonated eggs. Molecular detection of NDV was done by reverse transcriptase polymerase chain reaction (RT-PCR) encoding HN protein. RT-PCR for HN gene of NDV generated DNA fragments sized 503 bp, which were then sequenced using ABI Prism. The results have shown that virus isolates were mostly lentogenic and might contribute to outbreak in East Java, Indonesia. Based on this fact, NDV infected native chickens can act as reservoir and contribute to outbreak in the poultry. Our study provides baseline information on genetic characteristics of NDV circulating in East Java and serves as a basic work for further research.

Transient expression of fusion and hemagglutinin-neuraminidase epitopes of Newcastle disease virus in maize as a potent candidate vaccine.

PurposeNewcastle disease (ND) represents a major viral disease across the world which imposes high costs to poultry producers for vaccination. Hemagglutinin-neuraminidase (HN) and fusion (F) proteins are the major immunogenic epitopes of Newcastle disease virus and hence, have been the main targets for development of anti-ND vaccines. This paper reports transient expression of a synthetic gene composing of four tandem repeats of HN and three tandem repeats of F epitopes in maize leaves as initial step toward production of recombinant vaccine against ND.Materials and MethodsThe synthetic gene was cloned in pBI121 plasmid to yield an expression vector. The vector was sophisticated by the addition of AUG codon, polyhistidine-tag, tobacco mosaic virus omega sequence, stop codon, and restriction sites. Leaf transformation was conducted by the agroinfiltration method. Molecular detection assays including polymerase chain reaction, reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate transgene expression in infiltrated leaves of the corn plant.ResultsThe result obtained in this research revealed that the transgene was transcribed and translated in maize leaves only 48 hours after infiltration. In the second phase of the experiment, the expressed protein was injected into rabbits. The result of the ELISA assay indicated induction of immune response in the rabbits after injection with the heterologous protein.ConclusionThese results confirm the feasibility of agroinfiltration for transient gene expression of viral epitopes in monocot plants which naturally resist stable transformation by Agrobacterium tumefaciens. Practical implications of this finding are discussed in detail and some recommendations for future studies are proposed.

Spillover of bat borne Rubulavirus in Australian horses – Horses as sentinels for emerging infectious diseases

Background: Over 1000 horses are investigated annually for Hendra virus (HeV)-like illness, of which very few (<1%) test HeV positive. In Australia, in addition to HeV, other zoonotic viruses have affected horses including Australian bat lyssavirus, West Nile Virus (Kunjin), Murray Valley encephalitis virus and Ross River virus. In 1997, Menangle virus (MenPV), family Paramyxovirus, genus Rubulavirus, caused severe reproductive failure in pigs and influenza-like illness with rash in two piggery staff. MenPV has been isolated from Australian flying fox urine along with novel related rubulaviruses and HeV. We describe evidence of natural exposure to bat-borne rubulaviruses in Australian horses as well as seroconversion suggesting causality of severe respiratory illness. Methods and materials: Three-hundred-and-seventy-four horses were tested by a multiplex microsphere-based immunoassay (MIA) for IgG against MenPV nucleocapsid (N) protein and a subset also against Tioman virus (TioPV) N protein and MenPV hemagglutinin-neuraminidase (HN) protein. Confirmatory testing comprised immunofluorescence assay (IFA) on Vero cells infected with MenPV and related rubulaviruses. Results: Median fluorescence intensities (MFI) against MenPV N and a prior prevalence estimate of 20% were used in a Bayesian latent class model to determine appropriate cut-offs for positive test classification. Assay sensitivity was estimated assuming a specificity of both 95% and 99%. MFI reflecting potentially significant IgG to MenPV N protein was demonstrated in 34% (94/274) of horses with high perceived flying fox exposure (29% in QLD and 32% in NSW) whereas horses without plausible exposure recorded insignificant MFI. IFA confirmed antibodies to three of five related flying fox rubulaviruses tested (MenPV, Yeppoon virus and Grove virus). Case presentations: Two young-adult geldings developed severe acute respiratory illness in 2016 featuring obtunded demeanour, tachypnoea, tachycardia, congested/ hyperaemic mucous membranes, pyrexia and serous nasal discharge. Convalescent sera revealed a greater-than 10-fold increase in MFI to MenPV N (965 to 12,886). IFA confirmed antibodies to MenPV present in convalescent sera only. Conclusion: We highlight the potential of optimised syndromic surveillance for emerging zoonoses and One Health benefit of horses as sentinels of emerging infectious disease. Future research should determine the significance of rubulavirus spillover to horses and any associated human health risks.

Spillover of bat borne rubulavirus in Australian horses – Horses as sentinels for emerging infectious diseases

Background: Over 1000 horses are investigated annually for Hendra virus (HeV)-like illness, of which very few (<1%) test HeV positive. In Australia, in addition to HeV, other zoonotic viruses have affected horses including Australian bat lyssavirus, West Nile Virus (Kunjin), Murray Valley encephalitis virus and Ross River virus. In 1997, Menangle virus (MenPV), family Paramyxovirus, genus Rubulavirus, caused severe reproductive failure in pigs and influenza-like illness with rash in two piggery staff. MenPV has been isolated from Australian flying fox urine along with novel related rubulaviruses and HeV. We describe evidence of natural exposure to bat-borne rubulaviruses in Australian horses as well as seroconversion suggesting causality of severe respiratory illness. Methods and materials: Three-hundred-and-seventy-four horses were tested by a multiplex microsphere-based immunoassay (MIA) for IgG against MenPV nucleocapsid (N) protein and a subset also against Tioman virus (TioPV) N protein and MenPV hemagglutinin-neuraminidase (HN) protein. Confirmatory testing comprised immunofluorescence assay (IFA) on Vero cells infected with MenPV and related rubulaviruses. Results: Median fluorescence intensities (MFI) against MenPV N and a prior prevalence estimate of 20% were used in a Bayesian latent class model to determine appropriate cut-offs for positive test classification. Assay sensitivity was estimated assuming a specificity of both 95% and 99%. MFI reflecting potentially significant IgG to MenPV N protein was demonstrated in 34% (94/274) of horses with high perceived flying fox exposure (29% in QLD and 32% in NSW) whereas horses without plausible exposure recorded insignificant MFI. IFA confirmed antibodies to three of five related flying fox rubulaviruses tested (MenPV, Yeppoon virus and Grove virus). Case presentations: Two young-adult geldings developed severe acute respiratory illness in 2016 featuring obtunded demeanour, tachypnoea, tachycardia, congested/ hyperaemic mucous membranes, pyrexia and serous nasal discharge. Convalescent sera revealed a greater-than 10-fold increase in MFI to MenPV N (965 to 12,886). IFA confirmed antibodies to MenPV present in convalescent sera only. Conclusion: We highlight the potential of optimised syndromic surveillance for emerging zoonoses and One Health benefit of horses as sentinels of emerging infectious disease. Future research should determine the significance of rubulavirus spillover to horses and any associated human health risks.

Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus.

Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a “cold-chain” is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315–375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability.

Human parainfluenza virus fusion complex glycoproteins imaged in action on authentic viral surfaces.

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.

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

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Early Stage Development of a Newcastle Disease Vaccine Candidate in Corn.

Newcastle disease (ND) is a viral disease that causes labored breathing, periorbital oedema, and ataxia in the majority of avian species. The available vaccines against Newcastle disease virus (NDV) are limited, owing to their low reactivity and multiple dosage requirements. Plant-based machinery provides an attractive and safe system for vaccine production. In the current study, we attempted to express fusion (F) and hemagglutinin-neuraminidase (HN) proteins (the protective antigens against NDV) under constitutive 35S and seed-specific Zein promoters, respectively. Almost 2–7.1-fold higher expression of F gene mRNA in transgenic corn leaves and 8–28-fold higher expression of HN gene mRNA in transgenic corn seeds were observed, when the expression was analyzed by real-time PCR on a relative basis as compared to non-transgenic control plant material (Leaves and seeds). Similarly, 1.66 μg/ml of F protein in corn leaves, i.e., 0.5% of total soluble protein, and 2.4 μg/ml of HN protein in corn seed, i.e., 0.8% of total seed protein, were found when calculated through ELISA. Similar levels of immunological response were generated in chicks immunized through injection of E. coli-produced pET F and pET HN protein as in chickens orally fed leaves and seeds of maize with expressed immunogenic protein. Moreover, the detection of anti-NDV antibodies in the sera of chickens that were fed maize with immunogenic protein, and the absence of these antibodies in chickens fed a normal diet, confirmed the specificity of the antibodies generated through feeding, and demonstrated the potential of utilizing plants for producing more vaccine doses, vaccine generation at higher levels and against other infectious diseases.

High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines.

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

An electrochemical impedance sensor for monitoring of gallic acid inhibited neuraminidase activity of PPR HN protein

Peste des petits ruminants (PPR) virus has a surface glycoprotein, hemagglutinin neuraminidase (HN) with functional sites for cell attachment and neuraminidase (NA) activity. The neuraminidase activity of PPR virus (Sungri/96) was biochemically characterized with fluorometric assay, enzyme linked lectin assay (ELLA) and electrochemical impedance spectroscopy (EIS). Furthermore, the inhibitory effect of gallic acid on the virus neuraminidase activity was also evaluated in-vitro in B95a cells. Screen printed carbon electrode were used for the EIS assay. The activity of the viral neuraminidase was presented as the change in charge transfer resistance (Rct). The measurement of neuraminidase activity with EIS was found comparable to ELLA. Cell toxicity (CC50) concentration of gallic acid was found to be 61.0 ± 1.0 µM in B95a cells. A dose dependent effect of gallic acid on the virus neuraminidase activity was observed in both EIS and ELLA measurement. The anti PPRV activity of gallic acid was quantified by cell ELISA and 7.0 ± 1.0 µM was calculated concentration that has 50% viral inhibition in B95a cells. The gallic acid used in present study had neuraminidase inhibitory activity both in ELLA and EIS, furthermore it also reduces the virus propagation in vitro in B95a cells. For high throughput screening of anti-neuraminidase compounds including phytocompounds, electrochemical impedance spectroscopy could be vividly used.

Establishing a Robust Manufacturing Platform for Recombinant Veterinary Vaccines: An Adenovirus-Vector Vaccine to Control Newcastle Disease Virus Infections of Poultry in Sub-Saharan Africa.

Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of public health global policies in the context of “one world, one health” principles. Sub-Saharan Africa is frequently impacted by outbreaks of poultry diseases such as avian influenza and Newcastle Disease (ND). Here, an adenovirus-vectored vaccine technology platform is proposed for rapid adaptation to ND or other avian viral threats in the region. Ethiopian isolates of the Newcastle Disease virus (NDV) were subjected to sequence and phylogenetic analyses, enabling the construction of antigenically matched vaccine candidates expressing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. A cost-effective vaccine production process was developed using HEK293 cells in suspension and serum-free medium. Productive infection in bioreactors (1–3 L) at 2 × 106 cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5–6 × 108 TCID50/mL (approximately 1011VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 × 1010 TCID50 of the vectors, and full protection against a lethal NDV challenge was provided by the vector expressing the F antigen. These results consolidate the basis for a streamlined and scalable-vectored vaccine manufacturing process for deployment in low- and medium-income countries.

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.

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.