Phylogenetic lineage dynamics of global parainfluenza virus type 3 post-COVID-19 pandemic.

During the coronavirus disease 2019 (COVID-19) pandemic, outbreaks of parainfluenza virus type 3 (PIV-3) decreased due to infection control measures. However, a post-pandemic resurgence of PIV-3 has recently been observed. Nonetheless, the role of viral genetic epidemiology, possibly influenced by a genetic bottleneck effect, remains unexplored. We investigated the phylogenetic structure of the publicly available PIV-3 whole-genome and hemagglutinin-neuraminidase (HN) gene sequences spanning the last 65 years, including the COVID-19 pandemic. Sequences were retrieved from the nucleotide database of the National Center for Biotechnology Information using the search term "Human respirovirus 3." Sequence subsets covering all six genes of PIV-3 or the HN gene were designated as the whole-genome and HN surveillance data sets, respectively. Using these data sets, we constructed maximum-likelihood phylogenetic trees and performed a time-scaled analysis using a Bayesian SkyGrid coalescent prior. A total of 455 whole-genome and 1,139 HN gene sequences were extracted, revealing 10 and 11 distinct lineages, respectively, with >98% concurrence in lineage assignments. During the 2020 COVID-19 pandemic, only three single-lineage clusters were identified in Japan, Korea, and the USA. The inferred year of origin for PIV-3 was 1938 (1903-1963) for the whole-genome data set and 1955 (1930-1963) for the HN gene data set. Our study suggests that PIV-3 epidemics in the post-COVID era are likely influenced by a pandemic-driven bottleneck phenomenon and supports previous hypotheses suggesting s that PIV-3 originated during the early half of the 20th century.IMPORTANCEUsing publicly available parainfluenza virus type 3 (PIV-3) whole-genome sequences, we estimated that PIV-3 originated during the 1930s, consistent with previous hypotheses. Lineage typing and time-scaled phylogenetic analysis revealed that PIV-3 experienced a bottleneck phenomenon in Korea and the USA during the coronavirus disease 2019 pandemic. We identified the conservative hemagglutinin-neuraminidase gene as a viable alternative marker in long-term epidemiological studies of PIV-3 when whole-genome analysis is limited.

The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

The artificial amino acid change in the sialic acid-binding domain of the hemagglutinin neuraminidase of newcastle disease virus increases its specificity to HCT 116 colorectal cancer cells and tumor suppression effect.

BACKGROUND: Oncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects. METHODS: Sialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells. RESULTS: S519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence. CONCLUSION: These results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.

Protective effects of a novel chimeric virus-like particle vaccine against virulent NDV and IBDV challenge.

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

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.

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein.

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Antigenic variation in hemagglutinin-neuraminidase of Newcastle disease virus isolated from Tibet, China.

Vaccines are widely used to prevent Newcastle disease virus (NDV). Under the pressure of immunization, NDVs with mutations among epitopes of F and HN protein were isolated, which indicates that the efficiency of vaccine may decrease in terms of preventing emerged NDV. However, the lack of evidences to support whether these mutations contribute to antigenic mutation and immune escape in NDV leading to the controversy that the matched vaccine is more effective than the mismatched vaccine. In this study, a genotype VII velogenic NDV strain (C22) was isolated from a vaccinated farm in Tibet, China. We found that this strain was close to NDV from east China, but it had a specific mutation (K138R) in one epitope (131DYIGGIGKE139) of HN protein. This mutation might change the interaction between amino acids in stalk-head link region of HN protein and then induce the specific antibody to worse recognize the C22 strain, but it did not alter viral virulence and growth ability. Then, the C22 strain was attenuated via modification of the F protein cleavage site to generate a matched vaccine. Comparing to a mismatched vaccine (LaSota), this matched vaccine showed advantages in inhibiting viral shedding and tissue damage. However, both vaccines induced chicken to generate similar level of neutralizing antibodies against C22, C22mut (R138K) and LaSota. These results suggest that the epitope mutation is insufficient to help NDV escaping neutralizing antibodies of vaccinated chicken, supporting that the merits of NDV matched vaccine are not totally related to humoral immunity.

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.

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.

Subnanometer structure of an enveloped virus fusion complex on viral surface reveals new entry mechanisms.

Paramyxoviruses-including important pathogens like parainfluenza, measles, and Nipah viruses-use a receptor binding protein [hemagglutinin-neuraminidase (HN) for parainfluenza] and a fusion protein (F), acting in a complex, to enter cells. We use cryo-electron tomography to visualize the fusion complex of human parainfluenza virus 3 (HN/F) on the surface of authentic clinical viruses at a subnanometer resolution sufficient to answer mechanistic questions. An HN loop inserts in a pocket on F, showing how the fusion complex remains in a ready but quiescent state until activation. The globular HN heads are rotated with respect to each other: one downward to contact F, and the other upward to grapple cellular receptors, demonstrating how HN/F performs distinct steps before F activation. This depiction of viral fusion illuminates potentially druggable targets for paramyxoviruses and sheds light on fusion processes that underpin wide-ranging biological processes but have not been visualized in situ or at the present resolution.

Design, Synthesis, and Antiviral Evaluation of Sialic Acid Derivatives as Inhibitors of Newcastle Disease Virus Hemagglutinin-Neuraminidase: A Translational Study on Human Parainfluenza Viruses.

Global infections with viruses belonging to the Paramyxoviridae, such as Newcastle disease virus (NDV) or human parainfluenza viruses (hPIVs), pose a serious threat to animal and human health. NDV-HN and hPIVs-HN (HN hemagglutinin-neuraminidase) share a high degree of similarity in catalytic site structures; therefore, the development of an efficient experimental NDV host model (chicken) may be informative for evaluating the efficacy of hPIVs-HN inhibitors. As part of the broad research in pursuit of this goal and as an extension of our published work on antiviral drug development, we report here the biological results obtained with some newly synthesized C4- and C5-substituted 2,3-unsaturated sialic acid derivatives against NDV. All developed compounds showed high neuraminidase inhibitory activity (IC50 0.03-13 µM). Four molecules (9, 10, 23, 24) confirmed their high in vitro inhibitory activity, which caused a significant reduction of NDV infection in Vero cells, accompanied by very low toxicity.

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 Neu5Acα2-6Galß1-4GlcNAcß, Neu5Acα2-3Galß1-3GlcNacß1-3Galß1-4Glc, and Neu5Acα2-6Galß1-4GlcNAcß1-3Galß1-4Glc 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.

Evaluation of Newcastle disease virus LaSota strain attenuated by codon pair deoptimization of the HN and F genes for in ovo vaccination.

In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. The codon pair deoptimization (CPD) approach has been used to efficiently and rapidly attenuate viruses by targeting the virulence genes. In this study, we aimed to attenuate the NDV LaSota (LS) vaccine strain for in ovo vaccination by CPD of the fusion (F) or/and hemagglutinin-neuraminidase (HN) genes with approximately 44 % suboptimal codon substitutions. Three NDV LS recombinants expressing codon deoptimized F (rLS/F-d), HN (rLS/HN-d), or both genes (rLS/F+HN-d) were generated using reverse genetics technology. Biological assays showed that the CPD viruses retained similar hemagglutination activity and growth ability to the parental rLS virus. The CPD of the HN gene slightly attenuated the rLS/HN-d and rLS/F+HN-d viruses, whereas the CPD of the F gene marginally increased the rLS/F-d virus pathogenicity compared to rLS. Nevertheless, all three CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo inoculation of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken's hatch and survival rates. These results suggested that the CPD of the surface glycoprotein genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS strain.

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.

Characterization of Human Parainfluenza Virus Receptor Using Terminal Sialic Acid Linkage-Modified Cells.

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) are respiratory pathogen viruses that bind to terminal sialic acids of glycoconjugates on the cell surface hemagglutinin-neuraminidase glycoprotein. Sialic acid residues are linked to the galactose residue primarily by α2,3 or α2,6 linkages on the terminal of glycoprotein or glycolipids. One of the major determinants of pathogenicity or tissue tropism is virus binding or infection specificity for each sialyl linkage. Sialic linkage-modified human blood cells or mammalian cells that mainly have α2,3- or α2,6-linked sialic acid residues on the surface can be prepared by treatment with linkage-specific sialidases or sialyltransferases. These linkage-modified cells can be used in hemagglutination assays to estimate virus particles' binding specificity, hemadsorption assays to estimate virus glycoproteins' binding specificity, and virus infectivity assays. These methods contribute to identifying the specificity of sialic acid lineage recognition of the hPIV or other sialic acid-binding viruses.

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.

α2,3-Linked Sialic Acids Are the Potential Attachment Receptor for Shaan Virus Infection in MARC-145 Cells.

Shaan virus (ShaV), a novel species of the genus Jeilongvirus, family Paramyxoviridae, was isolated from an insectivore bat (Miniopterus schreibersii) in Korea in 2016. ShaV particles contain a hemagglutinin-neuraminidase (HN) glycoprotein in their envelope that allows the virus to target cells. Typically, diverse paramyxoviruses with HN glycoprotein are reported to interact predominantly with sialic acids, but there are no studies of receptors for ShaV. In this study, the identification of potential receptors for ShaV was demonstrated using sialidase treatments, glycan microarray, magnetic bead-based virus binding assay, and neuraminidase inhibitor treatments. Pretreatment of MARC-145 cells with sialidase, which cleaves α2,3-linked sialic acids, showed higher inhibition of viral infection than α2,6-linked-specific sialidase. These data were supported by the binding of ShaV to predominantly α2,3-linked sialylated glycans in the screening of sialyl linkage patterns through glycan microarray. To further confirm the direct interaction between ShaV and α2,3-linked sialic acids, ShaV was incubated with α2,3- or α2,6-linked sialylated glycans conjugated to magnetic beads, and binding signals were detected only for α2,3-linked sialylated glycans. In addition, the potential of sialic acids as a receptor was demonstrated by the viral replication inhibitory effect of the neuraminidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminicacid (DANA) in the mature virion release steps. Collectively, these results support that α2,3-linked sialic acids are the potential receptor for ShaV infection in MARC-145 cells. IMPORTANCE Bats host major mammalian paramyxoviruses, and novel paramyxoviruses are increasingly being reported around the world. Shaan virus (ShaV), from the genus Jeilongvirus, family Paramyxoviridae, has a potential attachment glycoprotein, HN. Here, we identify that ShaV binds to sialic acid and demonstrate that α2,3-linked sialic acids are the potential receptor for ShaV infection. The presented data regarding ShaV receptor specificity will enable studies into the viral tropism for the host and contribute to the development of new antiviral strategies targeting viral receptors.

Genetic Analysis of HPIV3 That Emerged during the SARS-CoV-2 Pandemic in Gwangju, South Korea.

Community mitigation measures taken owing to the COVID-19 pandemic have caused a decrease in the number of respiratory viruses, including the human parainfluenza virus type 3 (HPIV3), and a delay in their occurrence. HPIV3 was rarely detected as a consequence of monitoring respiratory viral pathogens in Gwangju, Korea, in 2020; however, it resurfaced as a delayed outbreak and peaked in September-October 2021. To understand the genetic characteristics of the reemerging virus, antigenic gene sequences and evolutionary analyses of the hemagglutinin-neuraminidase (HN) and fusion (F) genes were performed for 129 HPIV3 pathogens prevalent in Gwangju from 2018 to 2021. Unlike the prevalence of various HPIV3 strains in 2018-2019, the prevalence of HPIV3 by strains with reduced diversity was confirmed in 2021. It could be inferred that this decrease in genetic diversity was due to the restriction of inflow from other regions at home and abroad following the community mitigation measures and the spread within the region. The HPIV3 that emerged in 2021 consisted of HN coding regions that were 100% consistent with the sequence identified in Saitama, Japan, in 2018, and F coding regions exhibiting 99.6% homology to a sequence identified in India in 2017, among the ranks reported to the National Center for Biotechnology Information. The emergence of a new lineage in a community can lead to a mass outbreak by collapsing the collective immunity of the existing acquired area; therefore, continuous monitoring is necessary.

Quantitative regulation of the thermal stability of enveloped virus vaccines by surface charge engineering to prevent the self-aggregation of attachment glycoproteins.

The development of thermostable vaccines can relieve the bottleneck of existing vaccines caused by thermal instability and subsequent poor efficacy, which is one of the predominant reasons for the millions of deaths caused by vaccine-preventable diseases. Research into the mechanism of viral thermostability may provide strategies for developing thermostable vaccines. Using Newcastle disease virus (NDV) as model, we identified the negative surface charge of attachment glycoprotein as a novel determinant of viral thermostability. It prevented the temperature-induced aggregation of glycoprotein and subsequent detachment from virion surface. Then structural stability of virion surface was improved and virus could bind to and infect cells efficiently after heat-treatment. Employing the approach of surface charge engineering, thermal stability of NDV and influenza A virus (IAV) vaccines was successfully improved. The increase in the level of vaccine thermal stability was determined by the value-added in the negative surface charge of the attachment glycoprotein. The engineered live and inactivated vaccines could be used efficiently after storage at 37°C for at least 10 and 60 days, respectively. Thus, our results revealed a novel surface-charge-mediated link between HN protein and NDV thermostability, which could be used to design thermal stable NDV and IAV vaccines rationally.