Hemagglutinin-neuraminidase Protein Research Articles

Development of Hemagglutinin-Neuraminidase Homologous Peptides as Novel Promising Therapeutic Agents Against Peste des Petits Ruminants Virus.

The lack of specific antiviral therapy and complications associated with the existing peste des petits ruminants (PPR) vaccines accentuates the search of novel antiviral blocking agents in order to curtail the PPR infection at initial level. The synthetic hemagglutinin-neuraminidase (HN) homologous peptides may compete with the natural HN protein of PPR virus for binding to signaling lymphocytic activation molecule (SLAM) receptor, consequently, may disrupt peste des petits ruminants virus (PPRV) at entry level. Therefore, insilico analysis, synthesis, purification and subsequent characterization of HN homologous peptides were conducted in this study. The HN homologous peptides were synthesized by means of solid phase chemistry and were purified by reversed-phase-high performance liquid chromatography. The mass as well as sequence of HN homologous peptides were assessed by mass spectroscopy while its secondary structure was elucidated by circular dichroism spectroscopy. The binding (interaction) efficacy of HN homologous peptides with PPRV antibodies was assessed via indirect enzyme linked immunosorbent assay, visual detection test (red wine to purple), bathochromic shift under UV-Vis spectrophotometry and lateral flow immunochromatographic strip test. The antiviral properties and cytotoxicity of these peptides were also assessed in B95a cell line with changes in cytopathic effect and titer of PPRV (Sungri/96). The presence of green fluorescein isothiocyanate over the B95a cell surface pointed towards the binding of HN homologous peptides with surface SLAM receptor. Moreover, the intact beta sheet configuration in water and lower cytotoxicity [cytotoxic concentration 50 (CC50) > 1000µg/ml] of these peptides signifies its in vivo use. Among HN homologous peptides, the binding efficacy and antiviral properties of pep A was relatively high in comparison to pep B and Pep ppr peptides. The prerequisite concentration of HN homologous peptides (pep A = 12.5µg/ml; pep B = 25µg/ml; pep ppr = 25µg/ml) to exemplify its antiviral effect was much lower than its CC50 level. Hence, this study signifies the therapeutic potential of synthetic HN homologous peptides.

C-terminal truncation of the hemagglutinin-neuraminidase (HN) protein enhances the virulence and immunogenicity of Newcastle disease virus (NDV) vaccine strain V4.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein with receptor recognition ability that plays an important role in the infection of cells by NDV. An alignment of NDV HN protein sequences of different genotypes showed that vaccine strains of NDV, such as the LaSota strain, generally have an HN protein of 577 amino acids. In comparison, the HN protein of the V4 strain has 616 amino acids, with 39 more amino acids at the C-terminus. In this study, we generated a recombinant NDV (rNDV) with a 39-amino-acid truncation at the HN C-terminus based on the full-length cDNA clone of the V4 strain. This rNDV, named rV4-HN-tr, displayed thermostability similar to that of the parental V4 strain. However, growth kinetics and pathogenicity analysis suggested that rV4-HN-tr is more virulent than the V4 strain. Notably, the C-terminus of HN affected the ability of the virus to adsorb onto cells. Structural predictions further suggested that the C-terminus of HN may obstruct the sialic acid binding site. Immunization of chickens with rV4-HN-tr induced a 3.5-fold higher level of NDV-specific antibodies than that obtained with the V4 strain and provided 100% immune protection against NDV challenge. Our study suggests that rV4-HN-tr is a thermostable, safe, and highly efficient vaccine candidate against Newcastle disease.

In silico Prediction and Evaluation of Human Parainfluenza Virus-3 CD4+ T Cell Epitopes.

Human parainfluenza viruses type 3 (HPIV-3) through bronchiolitis and pneumonia is a common cause of lower respiratory tract infections. It is the main cause of hospitalization of infants and young children and also one of the main causes of morbidity and mortality in immuno-compromised and transplant patients. Despite many efforts, there is currently no specific anti-HPIV-3 drug or approved vaccine to prevent and control the virus. Identification of HPIV-3 epitopes with the capability of binding to human leukocyte antigen (HLA) class II molecules can be helpful in designing new vaccine candidates against HPIV-3 infection, and also can be useful for the in vitro stimulation and proliferation of HPIV-3-specific T cells for transplant and immunocompromised patients. To predict and comprehensively evaluate CD4+T cell epitope (HLA-II binders) from four main HPIV-3 antigens. In the present work, we predicted and comprehensively evaluated CD4+T cell epitope (HLA-II binders) from four main HPIV-3 antigens, including fusion protein (F), hemagglutininneuraminidase (HN), nucleocapsid (N) and matrix (M) proteins using bio- and immunoinformatics software. The toxicity, allergenicity, Blast screening and population coverage of the predicted epitopes were evaluated. The binding ability of the final selected epitopes was evaluated via a docking study. After several filtering steps, including blast screening, toxicity and allergenicity assay, population coverage and docking study, 9 epitopes were selected as candidate epitopes. The selected epitopes showed high population coverage and docking studies revealed a significantly higher binding affinity for the final epitopes in comparison with the negative control peptides. The final selected epitopes could be useful in designing vaccine candidates and for the treatment of immune-compromised individuals and patients with transplantation.

Molecular characterization and phylogenetic study of the hemagglutinin-neuraminidase gene of newcastle disease virus isolated from peacock (Pavo cristatus) and Turkey (Meleagris) and its comparison with broiler isolates.

Newcastle disease has been endemic within the Iranian poultry industry for decades. However, the genetic nature of the circulating Hemagglutinin-Neuraminidase (HN) gene among Iranian domesticated bird populations is broadly unexplored. The presented study was carried out to gain insights into the biological and molecular characterization of four complete HN genes isolated from turkey, peacock, and broiler isolates in Iran between 2018 and 2020. The phylogenetic analysis revealed that the isolates belong to the Newcastle disease virus (NDV) subgenotype VII.1.1, previously known as VIIL. Further analysis demonstrated the thermostable substitutions S315P and I369V within the isolates. Finding the N-glycosylation site (NIS) at positions 144-146 and the cysteine residue 123 might influence the fusogenicity abilities of the isolates, while identification of multiple amino acid substitutions in both antigenic sites, especially I514V and E347Q, and the binding sites of the HN protein, raised concern about the pathogenicity of the isolates. In addition, the annual rate of change based on the HN gene of Iranian NDV was calculated at about 1.8088E-3 between 2011 and 2020. In conclusion, a new NDV variant with multiple site mutagenesis is circulating not only among chickens but also in turkey and captive birds such as peafowls, and failure of routine vaccination programs could be attributed to the differences between circulating NDV strains and those used in vaccine manufacturing. Therefore, future legislation aimed at providing vaster vaccination cover and biosecurity plans will be needed to control the spread of circulating NDV strains.

Novel advances in parainfluenza virus 3 neutralization through antibodies targeting hemagglutinin-neuraminidase.

Abstract Parainfluenza virus 3 (PIV3) remains to be a global burden to infants, the elderly, and immunocompromised individuals. With no approved therapeutics or vaccines, PIV3 continues to cause a large portion of the lower respiratory illness hospitalizations in young children. Similar to other paramyxoviruses, PIV3 utilizes two structural proteins, the fusion (F) protein and the hemagglutinin-neuraminidase (HN) protein, for viral fusion and entry. Here, we characterize newly identified human monoclonal antibodies (mAbs) targeting HN on the PIV3 virion, with EC 50values ranging from 9ng/mL to 21ng/mL. The mAbs were tested for neutralization of PIV3 and all were determined to have an IC 50values ranging from 11ng/mL to 450ng/mL. Utilizing biolayer interferometry, we conducted a competitive epitope mapping study and found the mAbs targeted three major epitopes on the HN protein with some competitive binding between multiple sites, suggesting these epitopes are within close proximity of each other. mAbs binding to one particular site had significantly low IC 50values ranging from 11ng/mL to 120ng/mL. We have established a PIV3 infection model in Golden Syrian hamsters and are currently testing mAbs from each epitope for prevention of viral replication in the lungs and airways. Due to the similarities between the HN protein amongst different parainfluenza virus subtypes, we plan to study the potential cross-neutralizing capabilities of these mAbs against other parainfluenza virus subtypes. Supported by grants from NIH (R01 AI143865)

Potently neutralizing human mAbs against the zoonotic pararubulavirus Sosuga virus.

Sosuga virus (SOSV) is a recently discovered paramyxovirus with a single known human case of disease. There has been little laboratory research on SOSV pathogenesis or immunity, and no approved therapeutics or vaccines are available. Here, we report the discovery of human mAbs from the circulating memory B cells of the only known human case and survivor of SOSV infection. We isolated 6 mAbs recognizing the functional attachment protein hemagglutinin-neuraminidase (HN) and 18 mAbs against the fusion (F) protein. The anti-HN mAbs all targeted the globular head of the HN protein and could be organized into 4 competition-binding groups that exhibited epitope diversity. The anti-F mAbs can be divided into pre- or postfusion conformation-specific categories and further into 8 competition-binding groups. The only Ab in the panel that did not display neutralization activity was the single postfusion-specific anti-F mAb. Most of the anti-HN mAbs were more potently neutralizing than the anti-F mAbs, with mAbs in 1 of the HN competition-binding groups possessing ultrapotent (<1 ng/mL) half-maximal inhibitory virus neutralization values. These findings provide insight into the molecular basis for human Ab recognition of paramyxovirus surface proteins and the mechanisms of SOSV neutralization.

Molecular Evolution of Human Parainfluenza Virus Type 2 Based on Hemagglutinin-Neuraminidase Gene.

To understand the molecular evolution of human parainfluenza virus type 2 (HPIV2), 21 Hemagglutinin-Neuraminidase (HN) gene sequences covering seven Chinese provinces in 2011 and 2017 to 2021 were combined with 90 published HN sequences worldwide for phylogenetic analysis. The result showed that global HPIV2 could be classified into two distinct clusters (I and II), five lineages (IA to IIE), and four sublineages (IB1 and 2, and IIE1 and 2). The minimum genetic distances between different clusters and lineages were 0.049 and 0.014, respectively. In the last decade, one lineage (IID) and three sublineages (IB1, IB2, and IIE1) have been cocirculating in China, with the sublineages IB2 and IIE1 dominating, while sublineages IB1 and IIE1 are dominant globally. In addition, the spread of HPIV2 had relative spatial clustering, and sublineage IB2 has only been detected in China thus far. The overall evolution rate of HPIV2 was relatively low, on the order of 10-4 substitutions/site/year, except for sublineage IB2 at 10-3 substitutions/site/year. Furthermore, human-animal transmission was observed, suggesting that the HPIV2 might have jumped out of animal reservoirs in approximately 1922, the predicted time of a common ancestor. The entire HN protein was under purifying/negative selection, and the specific amino acid changes and two novel N-glycosylation sites (N316 and N517) in sublineages IB1, IB2, and IIE1 were mostly located in the globular head region of the HN protein. In this study, preliminary evolutionary characteristics of HPIV2 based on the HN gene were obtained, increasing the recognition of the evolution and adaptation of HPIV2. IMPORTANCE The phylogenetic analysis showed that global HPIV2 could be classified into two distinct clusters (I and II) and five lineages (IA to IIE) with at least 0.049 and 0.014 genetic distances between clusters and lineages, respectively. Furthermore, lineages IB and IIE could be further divided into two sublineages (IB1-2 and IIE1-2). All China sequences belong to one lineage and three sublineages (IB1, IB2, IID, and IIE1), among which sublineages IB2 and IIE1 are predominant and cocirculating in China, while sublineages IB1 and IIE1 are dominant globally. The overall evolution rate of HPIV2 is on the order of 10-4 substitutions/site/year, with the highest rate of 2.18 × 10-3 for sublineage IB2. The entire HN protein is under purifying/negative selection, and the specific amino acid substitutions and two novel N-glycosylation sites (N316 and N517) in sublineages IB1, IB2, and IIE1 are mostly located in the globular head region of the HN protein.

Kinetic analysis of paramyxovirus-sialoglycan receptor interactions reveals virion motility.

Many viruses initiate infection by binding to sialoglycan receptors at the cell surface. Binding to such receptors comes at a cost, however, as the sheer abundance of sialoglycans e.g. in mucus, may immobilize virions to non-functional decoy receptors. As a solution, sialoglycan-binding as well as sialoglycan-cleavage activities are often present in these viruses, which for paramyxoviruses are combined in the hemagglutinin-neuraminidase (HN) protein. The dynamic interactions of sialoglycan-binding paramyxoviruses with their receptors are thought to be key determinants of species tropism, replication and pathogenesis. Here we used biolayer interferometry to perform kinetic analyses of receptor interactions of animal and human paramyxoviruses (Newcastle disease virus, Sendai virus, and human parainfluenza virus 3). We show that these viruses display strikingly different receptor interaction dynamics, which correlated with their receptor-binding and -cleavage activities and the presence of a second sialic acid binding site. Virion binding was followed by sialidase-driven release, during which virions cleaved sialoglycans until a virus-specific density was reached, which was largely independent of virion concentration. Sialidase-driven virion release was furthermore shown to be a cooperative process and to be affected by pH. We propose that paramyxoviruses display sialidase-driven virion motility on a receptor-coated surface, until a threshold receptor density is reached at which virions start to dissociate. Similar motility has previously been observed for influenza viruses and is likely to also apply to sialoglycan-interacting embecoviruses. Analysis of the balance between receptor-binding and -cleavage increases our understanding of host species tropism determinants and zoonotic potential of viruses.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

On the potentials of sialic acid derivatives as inhibitors for the mumps virus: A molecular dynamics and quantum chemistry investigation

Mumps virus is an infectious pathogen causing major health problems for humans such as encephalitis, orchitis, and parotitis. Therefore, designing an inhibitor for this virus is of great medical and public health importance. With this goal in mind, we investigate the affinity of different sialic acid-based compounds (ligands) against the hemagglutinin-neuraminidase (HN) protein of the mumps virus, using a combination of molecular dynamics (MD) simulations and quantum chemistry calculations. Our MD simulation results indicate that the ligands form stable complexes with the HN protein through a combination of electrostatic, van der Waals (vdW), and hydrogen bond (H-bond) interactions, which the electrostatic interactions play a more important role in the complexation process. Based on the obtained results from the structural analysis Arg381, Arg291, and Arg49 play a key role in the binding site interactions with the different ligands, in comparison with other residues. There are some candidates such as Neu5Acα2-6Galβ1-4GlcNAcβ, Neu5Acα2-3Galβ1-3GlcNacβ1-3Galβ1-4Glc, and Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc that form more stable complexes with the HN than the α2-3-Sialyllactose confirmed by the calculated Gibbs binding energies (-39.65, -46.93, and -36.49 kcal.mol−1, respectively).To investigate the relationship between the molecular properties of the selected compounds and their affinity to the HN receptor, density functional theory dispersion corrected (DFT-D3) calculations were employed. According to our DFT-D3 results, neutral sialic acid-based compounds have lower reactivity to the mumps virus than the negativity charge structures. Moreover, by increasing the electronic chemical potential (μ) the vdW and H-bond interactions between drugs and the HN protein increase. In other words, by elevating the electron tendency of the selected ligands their affinity to the mumps virus increases. Our quantum chemistry calculations reveal that in addition to the structural features the molecular properties of the drugs can play important roles in their affinity and reactivity against the virus. The results of this study can provide useful details to design new compounds or improve their properties against the mumps virus.

Abstract 1256: The surface-specific antigenic peptide designs of HPIV-type 2 Hemagglutinin-Neuraminidase and Fusion protein for anti-viral immunotherapy drug development

Abstract 1256: The surface-specific antigenic peptide designs of HPIV-type 2 Hemagglutinin-Neuraminidase and Fusion protein for anti-viral immunotherapy drug development

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells.

Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms. The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle. Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication. These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.

Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran.

Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.

Biological Significance of Dual Mutations A494D and E495K of the Genotype III Newcastle Disease Virus Hemagglutinin-Neuraminidase In Vitro and In Vivo.

As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.

Epicatechin analogues may hinder human parainfluenza virus infection by inhibition of hemagglutinin neuraminidase protein and prevention of cellular entry.

Human parainfluenza viruses (HPIVs) are ( -)ssRNA viruses belonging to Paramyoviridaie family. They are one of the leading causes of mortality in infants and young children and can cause ailments like croup, bronchitis, and pneumonia. Currently, no antiviral medications or vaccines are available to effectively treat parainfluenza. This necessitates the search for a novel and effective treatment. Computer-aided drug design (CADD) methodology can be utilized to discover target-based inhibitors with high accuracy in less time. A library of 45 phytocompounds with immunomodulatory properties was prepared. Thereafter, molecular docking studies were conducted to characterize the binding behavior of ligand in the binding pocket of HPIV3 HN protein. The physicochemical properties for screened compounds were computed, and the top hits from docking studies were further analyzed and validated using molecular dynamics simulation studies using the Desmond module of Schrodinger Suite 2021-1, followed by MM/GBSA analysis. The compounds CID:72276 (1) and CID:107905 (2) emerged as lead compounds of our in silico investigation. Further in vitro studies will be required to prove the efficacy of lead compounds as inhibitors and to determine the exact mechanism of their inhibition. Computational studies predict three natural flavonoids to inhibit the HN protein of HPIV3.

Development of Novel Recombinant Antigens of Nucleoprotein and Matrix Proteins of Porcine orthorubulavirus: Antigenicity and Structural Prediction.

Blue eye disease (BED) is a swine viral infection that affects the pork industry of Mexico. Porcine orthorubulavirus (PRV) is the etiological agent, and the hemagglutinin-neuraminidase protein (HN) is characterized as the best antigen for serological tests, although other structural proteins, including the nucleoprotein (NP) and the matrix (M) protein, have been investigated during the infection of members of the Paramyxoviridae family, generating promising results. Herein, for the first time, we successfully produced and characterized both the NP and M proteins of PRV by using a recombinant strategy in the E. coli heterologous system. The ORF of the NP and M genes were cloned in-frame with the pET-SUMO expression vector. Recombinant proteins proved to be a sensitive target to detect seroconversion at 7 days until 28 days in vaccinated mice (BALB/c) by indirect ELISAs. Immunoreactivity was also tested using porcine serum samples, in which antibodies were recognized from early stages to a persistence of PRV infection, which is indicative that these proteins contain properties similar to native antigens. The predicted tertiary structure showed that both proteins have a conserved structure that resembles those found in others Paramyxovirus. Our results pave the way for developing biotechnological tools based on these proteins for the control and prevention of BED.

Analysis of mutations in the fusion protein and hemagglutinin-neuraminidase in twelve strains of NDV isolated in Madagascar by bioinformatics methods

Many researchers have made efforts to generate genotype-matched and subunit vaccines for Newcastle disease (ND). During the design process, we found that they only considered certain characteristics of the virulent strains. As a result, the resulting vaccines are still poorly effective against new emerging strains of ND virus (NDV). This analysis suggests that consideration of various characteristics of virulent strains in the design is necessary to increase the efficacy of these new vaccines. Thus, analysis of mutations in viral proteins targeted in the design such as fusion proteins (F) and hemagglutinin-neuraminidase (HN) is essential. For this reason, the present study aims to analyze mutations in these two proteins in twelve NDV strains isolated in Madagascar. To achieve this, we employed bioinformatics methods such as sequence alignment, molecular modeling, 3D structure comparison and mutation impact prediction via bioinformatics software and servers. As results, we identified respectively 26 and 41 mutations in the F and HN proteins of the Madagascar isolates. All these mutations have an impact on the stability of the protein. However, only 6 mutations (D344N, G303V, P315S, E347K, P391S and E495S) have an impact on their 3D structures. Finally, the 3 mutations (A477T, G303V and P315S) also affect the functions of these two proteins. In conclusion, we have identified 67 mutations that may affect the stability, structures, and functions of NDV F and HN proteins. Further studies are needed to know their effects on the antigenicity and immunogenicity of these two proteins.

First Isolation and Characteristics of Bovine Parainfluenza Virus Type 3 from Yaks.

The yaks belong to the genus Bos within the family Bovidae that live in the Tibet Plateau and is an indispensable economic resource for the local herders. Respiratory tract infections are common diseases in yaks caused by various pathogens; however, there have been no reports of bovine parainfluenza virus type 3 (BPIV3) infection. This study was conducted to investigate the pathogens and analyze their characteristics from the four yak lung samples with severe respiratory tract infection symptoms in the yak farm. Results showed that out of four lung samples, three were identified as BPIV3-positive by RT-PCR. A BPIV3 strain (106.5 TCID50/mL) was successfully isolated from the BPIV3-positive lung samples using Madin–Darby bovine kidney cells. The isolate caused systemic infection in the BALB/c mice and induced pathological changes in the lungs. Moreover, three complete BPIV3 genomes were amplified from the clinical samples. Phylogenetic trees based on the complete genomes, hemagglutinin-neuraminidase protein (HN), phosphoprotein (P), and large polymerase subunit protein (L) amino acid sequences showed that the complete BPIV3 genomes belonged to BPIV3 genotype C, and clustered into a large branch with the Chinese strains, although the three yak BPIV3 strains were clustered into a small branch. Compared to known BPIV3 genotype C strains in GenBank, the three genomes of yak BPIV3 showed four identical amino acid mutations in the HN, P and L proteins, suggesting a unique genetic evolution of BPIV3 in yaks. This study first isolated and characterized the BPIV3 from yaks, which contributed to the understanding of the infection and evolution of BPIV3 in yaks in the Tibet Plateau.

Execution and Design of an Anti HPIV-1 Vaccine with Multiple Epitopes Triggering Innate and Adaptive Immune Responses: An Immunoinformatic Approach.

Human Parainfluenza Virus (HPIV) Type-1, which is an anti-sense ribonucleic acid (RNA) virus belonging to the paramyxoviridae family, induces upper and lower respiratory tract infections. The infections caused by the HPIV Type-1 virus are usually confined to northwestern regions of America. HPIV-1 causes infections through the virulence of the hemagglutinin-neuraminidase (HN) protein, which plays a key role in the attachment of the viral particle with the host’s receptor cells. To the best of our knowledge, there is no effective antiviral drugs or vaccines being developed to combat the infection caused by HPIV-1. In the current study, a multiple epitope-based vaccine was designed against HPIV-1 by taking the viral HN protein as a probable vaccine candidate. The multiple epitopes were selected in accordance with their allergenicity, antigenicity and toxicity scoring. The determined epitopes of the HN protein were connected simultaneously using specific conjugates along with an adjuvant to construct the subunit vaccine, with an antigenicity score of 0.6406. The constructed vaccine model was docked with various Toll-like Receptors (TLRs) and was computationally cloned in a pET28a (+) vector to analyze the expression of vaccine sequence in the biological system. Immune stimulations carried out by the C-ImmSim Server showed an excellent result of the body’s defense system against the constructed vaccine model. The AllerTop tool predicted that the construct was non-allergen with and without the adjuvant sequence, and the VaxiJen 2.0 with 0.4 threshold predicted that the construct was antigenic, while the Toxinpred predicted that the construct was non-toxic. Protparam results showed that the selected protein was stable with 36.48 instability index (II) scores. The Grand average of Hydropathicity or GRAVY score indicated that the constructed protein was hydrophilic in nature. Aliphatic index values (93.53) confirmed that the construct was thermostable. This integrated computational approach shows that the constructed vaccine model has a potential to combat laryngotracheobronchitis infections caused by HPIV-I.

Amino Acid Mutations in Hemagglutinin-Neuraminidase Enhance the Virulence and Pathogenicity of the Genotype III Newcastle Disease Vaccine Strain After Intravenous Inoculation

Newcastle disease virus (NDV), the causative agent that generally causes severe disease in poultry, continues to mutate and has thus evolved into 21 genotypes. We previously isolated a velogenic genotype III NDV JS/7/05/Ch that evolved from the vaccine strain Mukteswar, accompanying by amino acid mutations in Hemagglutinin-Neuraminidase (HN). Here, we sought to investigate the role of the mutant HN protein in NDV virulence. The HN genes of Mukteswar and JS/7/05/Ch were replaced reciprocally via reverse genetics, yielding two recombinant viruses rJS/MHN and rMu/JHN, respectively. rMu/JHN, in which the endogenous HN protein was replaced with the HN protein of JS/7/05/Ch, had a higher intravenous pathogenicity index (IVPI) value in chickens. Moreover, dual aa mutations (A494D and E495K from JS/7/05/Ch-type HN) were introduced into the HN protein of Mukteswar to generate the recombinant virus rMukHN494+495JS. This virus showed an equivalent IVPI value to that of rJS/7/05/Ch (generated from parental JS/7/05/Ch via reverse genetics). In vitro and in vivo assays further showed that A494D and E495K in HN induced antigenic changes, a higher replication level and a more intense inflammatory response. Taken together, these findings indicate that aa mutations in HN are crucial for the virulence of the genotype III Newcastle disease (ND) vaccine strain after intravenous inoculation. Our study further highlights that close surveillance is needed to monitor the genetic variation of ND vaccine strains.