M2 Protein Research Articles

M2e-Derived Peptidyl and Peptide Amphiphile Micelles as Novel Influenza Vaccines

Background: A significant problem with current influenza vaccines is their reliance on predictions of the most prevalent strains for the upcoming season, with inaccurate forecasts greatly reducing the overall efficacy of the immunization campaign. A universal influenza vaccine, which leverages epitopes conserved across many, if not all, strains of influenza, could reduce the need for extremely accurate forecasting. The highly conserved ectodomain of the influenza M2 protein contains a B cell epitope in the M22–16 region, making it a promising candidate as a universal influenza vaccine. Unfortunately, free peptide antigens alone are limited as vaccines due to their poor stability and weak immunogenicity in vivo. To improve the potential of peptide vaccines, immunostimulatory micellar nanoparticles can be generated from them by lipid conjugation (i.e., peptide amphiphiles—PAs). Methods: M22–16 peptides and Palm2K-M22–16-(KE)4 PAs were synthesized and characterized. BALB/c mice were subcutaneously vaccinated with these formulations, and ELISAs were conducted on serum collected from the vaccinated mice to evaluate induced antibody responses. Results: Unlike other peptide antigens previously studied, the unmodified M22–16 peptide micellized without any peptidyl or lipid modifications. M22–16 peptidyl micelles (PMs) were spherical with largely undefined secondary structure somewhat different from the cylindrical, β-sheet-containing Palm2K-M22–16-(KE)4 peptide amphiphile micelles (PAMs). Differences in physical properties were found to correlate with slightly different immune responses with PAMs eliciting higher antibody titers after the initial immunization, whereas both micelle types elicited strong IgG titers after a prime-boost regimen. Conclusions: These results suggest the viability of PAMs as single-dose vaccines, while both PMs and PAMs show potential using a multi-dose immunization approach.

Seeing double: the persistent dimer-of-dimers structure of drug resistant influenza A M2.

The currently circulating S31N variant of the M2 proton channel of influenza A is resistant to antiviral drugs. Recently, there has been a growing concern regarding the impact of the lipid environment on the structural features of the S31N variant. The native symmetry of the M2 tetramer remains controversial. Here we show that S31N M2 persists in a dimer-of-dimers structure in different lipid preparations independent of the amount of solvating lipids up to at least 180 lipids per tetramer. NMR spectra clearly detect the characteristic resonances of the dimer-of-dimers of M2 (residues 18-60 or 18-62) reconstituted in lipids. NMR-based distance measurements indicate that two isoleucine residues with upfield shifted alpha carbon resonances, typical of extended conformations, are compatible with a particular side-chain rotameric state and helical backbone geometry. These chemical shifts are therefore compatible with the expected native transmembrane helical fold. Symmetry breaking at the pH sensing H37 residues, detected via peak doubling, is a stable feature of S31N M2 based on the reference strain Udorn/1972(H3N2). By contrast, the spectrum is dramatically altered for Columbia/2014/(H3N2) M2, which differs in sequence in the amphipathic helices. This highlights an allosteric coupling between the amphipathic helices and the pH sensing residues.

Stable and Minimum Size Solubilization of Membrane Proteins with Cocktails of Phospholipid Analogues.

Membrane proteins (MPs) play important roles in various cellular processes and are major targets for drugs. Solubilization of MPs is often needed for structural and biophysical studies. For high-resolution nuclear magnetic resonance measurements, there is a size limit of the sample (<100 kDa), and a high thermal stability at an increased temperature is required. Furthermore, lipid bilayer-like environments are desirable to preserve the native states of MPs. However, existing solubilization techniques do not fulfill these requirements at the same time. In this study, we combined two phospholipid analogues as a solubilizer and stabilizer to isolate MPs. This method maintained bacteriorhodopsin (bR) extracted from purple membranes in its native state for 7 d at 40 °C. The solubility was comparable to that of conventional detergents for MPs, and the thermal stability of the solubilizate was the best among them. The increase in the molecular size caused by the solubilization of bR was only 20 kDa, indicating that 20 phospholipid analogue molecules were sufficient to solubilize one bR molecule. 15N-1H heteronuclear single quantum coherence spectra of solubilized 2H- and 15N-labeled bR gave ∼80% of the expected peaks. In addition, the lysate of human neuropeptide Y2 receptor-expressing mammalian cells exhibited ligand recognition for 7 d at 37 °C, suggesting that this technique can be used for ligand screening. Moreover, the structure of the single membrane-spanning M2 protein of the influenza A virus expressed in Escherichia coli was stably maintained for 7 d at 40 °C. Thus, our method is promising for various MP studies.

Activation of the Influenza B M2 Proton Channel (BM2).

Influenza B viruses have cocirculated during most seasonal flu epidemics and can cause significant human morbidity and mortality due to their rapid mutation, emerging drug resistance, and severe impact on vulnerable populations. The influenza B M2 proton channel (BM2) plays an essential role in viral replication, but the mechanisms behind its symmetric proton conductance and the involvement of a second histidine (His27) cluster remain unclear. Here we performed membrane-enabled continuous constant-pH molecular dynamics simulations on wildtype BM2 and a key H27A mutant channel to explore its pH-dependent conformational switch. Simulations captured the activation as the first histidine (His19) protonates and revealed the transition at lower pH values compared to AM2 is a result of electrostatic repulsions between His19 and preprotonated His27. Crucially, we provided an atomic-level understanding of the symmetric proton conduction by identifying preactivating channel hydration in the C-terminal portion. This research advances our understanding of the function of BM2 function and lays the groundwork for further chemically reactive modeling of the explicit proton transport process as well as possible antiflu drug design efforts.

Epidemiological and Genetic Characterization of Human Infection with Avian Influenza AI H5N6 Virus in Guangxi, China, 2021

Avian influenza (AI) H5N6 has replaced H5N1 as the predominant strain, and the increasing number of cases and complex recombination patterns pose significant threats to human health. The data were collected on human cases of AI H5N6 from the National Surveillance of Notifiable Infectious Disease Programme (NSNIDP) in Guangxi and used MEGA software to conduct phylogenetic analysis with published reference strains obtained from the GISAID database. Between January 1 and December 31, 2021, 11 cases of human AI H5N6 infection were reported in Guangxi. All HA genes belonged to clade 2.3.4.4, with only one virus belonging to 2.3.4.4h and the remaining viruses falling into 2.3.4.4b. Phylogenetic analysis of the genes was conducted, and some substitutions were detected in the HA gene, M1 protein, and NS1 protein. A close relationship between Guangxi viruses and AIVs of poultry origin has been observed. Given the contamination of live poultry markets and backyard poultry, AI H5N6 variants and genotypes will continuously emerge; therefore, enhancing surveillance is crucial for pandemic preparedness.

Designing a multi-epitope influenza vaccine: an immunoinformatics approach

Influenza continues to be one of the top public health problems since it creates annual epidemics and can start a worldwide pandemic. The virus’s rapid evolution allows the virus to evade the host defense, and then seasonal vaccines need to be reformulated nearly annually. However, it takes almost half a year for the influenza vaccine to become accessible. This delay is especially concerning in the event of a pandemic breakout. By producing the vaccine through reverse vaccinology and phage display vaccines, this time can be reduced. In this study, epitopes of B lymphocytes, cytotoxic T lymphocytes, and helper T lymphocytes of HA, NA, NP, and M2 proteins from two strains of Influenza A were anticipated. We found two proper epitopes (ASFIYNGRL and LHLILWITDRLFFKC) in Influenza virus proteins for CTL and HTL cells, respectively. Optimal epitopes and linkers in silico were cloned into the N-terminal end of M13 protein III (pIII) to create a multi-epitope-pIII construct, i.e., phage display vaccine. Also, prediction of tertiary structure, molecular docking, molecular dynamics simulation, and immune simulation were performed and showed that the designed multi-epitope vaccine can bind to the receptors and stimulate the immune system response.

In Silico evaluation of the anti-influenza potential of Streptomyces bioactive compounds

Influenza, a highly contagious respiratory viral disease characterized by its short incubation period and severe symptoms, is treatable with antiviral drugs targeting viremia. Among new antiviral agents, Actinobacteria, particularly from the Streptomyces genus, are notable for their ability to produce potent antiviral compounds. This study evaluates the antiviral potential of Streptomyces sp. KSF 103, isolated from Kuala Sat, Jerantut, Pahang, against the Influenza A virus (IAV). Using molecular docking tools such as AutoDock Vina and PyMOL, the interactions of four compounds – hypoxanthine, vitamin D, purine, and aminocaproic acid – were analyzed against IAV proteins. Vitamin D exhibited the highest binding affinity against H3N2’s M2 (−9.6 kcal/mol) and HA (−9.2 kcal/mol) proteins. Additionally, RMSD dynamics parameters indicated that the best-scoring complexes are predicted to be satisfactorily stable under physiological settings. These findings suggest that Streptomyces sp. KSF 103 could be a promising source for developing new antiviral treatments against influenza.

Potent Anti-Influenza Synergistic Activity of Theobromine and Arainosine.

Influenza represents one of the biggest health threats facing humanity. Seasonal epidemics can transition to global pandemics, with cross-species infection presenting a continuous challenge. Although vaccines and several anti-viral options are available, constant genetic drifts and shifts vitiate any of the aforementioned prevention and treatment options. Therefore, we describe an approach targeted at the virus's channel to derive new anti-viral options. Specifically, Influenza A's M2 protein is a well-characterized channel targeted for a long time by aminoadamantane blockers. However, widespread mutations in the protein render the drugs ineffective. Consequently, we started by screening a repurposed drug library against aminoadamantane-sensitive and resistant M2 channels using bacteria-based genetic assays. Subsequent in cellulo testing and structure-activity relationship studies yielded a combination of Theobromine and Arainosine, which exhibits stark anti-viral activity by inhibiting the virus's channel. The drug duo was potent against H1N1 pandemic swine flu, H5N1 pandemic avian flu, aminoadamantane-resistant and sensitive strains alike, exhibiting activity that surpassed Oseltamivir, the leading anti-flu drug on the market. When this drug duo was tested in an animal model, it once more outperformed Oseltamivir, considerably reducing disease symptoms and viral RNA progeny. In conclusion, the outcome of this study represents a new potential treatment option for influenza alongside an approach that is sufficiently general and readily applicable to other viral targets.

Continuing evolution of H5N1 highly pathogenic avian influenza viruses of clade 2.3.2.1a G2 genotype in domestic poultry of Bangladesh during 2018–2021

ABSTRACT We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018–2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020–2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

Inserting CTL Epitopes of the Viral Nucleoprotein to Improve Immunogenicity and Protective Efficacy of Recombinant Protein against Influenza A Virus.

Conserved influenza virus proteins, such as the hemagglutinin stem domain (HA2), nucleoprotein (NP), and matrix protein (M), are the main targets in the development of universal influenza vaccines. Previously, we constructed a recombinant vaccine protein Flg-HA2-2-4M2ehs containing the extracellular domain of the M2 protein (M2e) and the aa76-130 sequence of the second HA subunit as target antigens. It demonstrated immunogenicity and broad protection against influenza A viruses after intranasal and parenteral administration. This study shows that CD8+ epitopes of NP, inserted into a flagellin-fused protein carrying M2e and HA2, affect the post-vaccination immune humoral response to virus antigens without reducing protection. No differences were found between the two proteins in their ability to stimulate the formation of follicular Th in the spleen, which may contribute to a long-lasting antigen-specific humoral response. The data obtained on Balb/c mice suggest that the insertion of CTL NP epitopes into the flagellin-fused protein carrying M2e and HA2 reduces the antibody response to M2e and A/H3N2. In C57Bl6 mice, this stimulates the formation of NP-specific CD8+ Tem and virus-specific mono- and multifunctional CD4+ and CD8+ Tem in the spleen and completely protects mice from influenza virus subtypes A/H1N1pdm09 and A/H3N2.

Genetic characteristics of influenza A and B viruses circulating in Russia in 2019-2023.

Relevance. Influenza viruses have a high potential for genetic change. These viruses are monitored annually around the world, including Russia, to determine the dominant genetic groups and select the strains to be included in influenza vaccines. Aims of the study include: analysis of the circulation of influenza viruses in Russia in 2019-2023, phylogenetic and molecular analysis of hemagglutinin (HA) sequences of influenza virus strains, detection of drug resistance mutations to neuraminidase (NA) inhibitors and M2-protein (M2) ion channel inhibitors. Materials and methods. Biological samples containing RNA of influenza viruses were studied: 417 A(H1N1)pdm09, 147 A(H3N2) and 167 B(Victoria). Sequencing of the PCR fragments was performed on the 3500xL Genetic Analyzer (Applied Biosytems). Data processing and analysis were carried out using DNASTAR, Nextclade, FluSurver and BioNumerics v.6.6 software. Results. Influenza A(H1N1)pdm09, A(H3N2), B(Victoria) viruses circulating in 2019-2023 were investigated. The highest variability of HA was observed in A(H3N2) viruses. 3% of A(H1N1)pdm09 viruses had the D222N mutation in the receptor-binding site of HA, which is associated with more severe disease. The oseltamivir and zanamivir resistance mutation H275Y in NA was detected in 2% of influenza A(H1N1)pdm09 viruses. No oseltamivir and zanamivir resistance mutations in NA were detected in all influenza A(H3N2) and B viruses tested. In all investigated influenza A(H1N1)pdm09 and A(H3N2) viruses the adamantane resistance mutation S31N in M2 was identified. Conclusions. The detection of amino acid substitutions in HA antigenic sites and resistance mutations in NA and M2 confirms the evolution of influenza viruses and the need for continuous genetic surveillance.

The selective autophagic degradation of avian metapneumovirus subgroup C M2-2 protein via SQSTM1 suppresses viral replication

The selective autophagic degradation of avian metapneumovirus subgroup C M2-2 protein via SQSTM1 suppresses viral replication

Influenza A Vaccine Candidates Based on Virus-like Particles Formed by Coat Proteins of Single-Stranded RNA Phages Beihai32 and PQ465.

Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum recombinant vaccines based on conserved antigens. The extracellular domain of the transmembrane protein M2 of influenza A virus (M2e) is highly conserved but poorly immunogenic and needs to be fused to an adjuvant protein or carrier virus-like particles (VLPs) to increase immunogenicity and provide protection against infection. In this study, we obtained VLPs based on capsid proteins (CPs) of single-stranded RNA phages Beihai32 and PQ465 bearing the M2e peptides. Four copies of the M2e peptide were linked to the C-terminus of the CP of phage Beihai32 and to the N and C termini of the CP of phage PQ465. The hybrid proteins, being expressed in Escherichia coli, formed spherical VLPs of about 30 nm in size. Immunogold transmission electron microscopy showed that VLPs formed by the phage PQ465 CP with a C-terminal M2e fusion present the M2e peptide on the surface. Subcutaneous immunization of mice with VLPs formed by both CPs containing four copies of the M2e peptide at the C termini induced high levels of M2e-specific IgG antibodies in serum and provided mice with protection against lethal influenza A virus challenge. In the case of an N-terminal fusion of M2e with the phage PQ465 CP, the immune response against M2e was significantly lower. CPs of phages Beihai32 and PQ465, containing four copies of the M2e peptide at their C termini, can be used to develop recombinant influenza A vaccine.

Bioinformatics Analysis Screening and Identification of Key Biomarkers and Drug Targets in Human Glioblastoma.

Glioblastoma is the most common type of brain cancer, with a prognosis that is unfortunately poor. Despite considerable progress in the field, the intricate molecular basis of this cancer remains elusive. The aim of this study was to identify genetic indicators of glioblastoma and reveal the processes behind its development. The advent and integration of supercomputing technology have led to a significant advancement in gene expression analysis platforms. Microarray analysis has gained recognition for its pivotal role in oncology, crucial for the molecular categorization of tumors, diagnosis, prognosis, stratification of patients, forecasting tumor responses, and pinpointing new targets for drug discovery. Numerous databases dedicated to cancer research, including the Gene Expression Omnibus (GEO) database, have been established. Identifying differentially expressed genes (DEGs) and key genes deepens our understanding of the initiation of glioblastoma, potentially unveiling novel markers for diagnosis and prognosis, as well as targets for the treatment of glioblastoma. This research sought to discover genes implicated in the development and progression of glioblastoma by analyzing microarray datasets GSE13276, GSE14805, and GSE109857 from the GEO database. DEGs were identified, and a function enrichment analysis was performed. Additionally, a protein-protein interaction network (PPI) was constructed, followed by module analysis using the tools STRING and Cytoscape. The analysis yielded 88 DEGs, consisting of 66 upregulated and 22 downregulated genes. These genes' functions and pathways primarily involved microtubule activity, mitotic cytokinesis, cerebral cortex development, localization of proteins to the kinetochore, and the condensation of chromosomes during mitosis. A group of 27 pivotal genes was pinpointed, with biological process analysis indicating significant enrichment in activities, such as division of the nucleus during mitosis, cell division, maintaining cohesion between sister chromatids, segregation of sister chromatids during mitosis, and cytokinesis. The survival analysis indicated that certain genes, including PCNA clamp-associated factor (PCLAF), ribonucleoside- diphosphate reductase subunit M2 (RRM2), nucleolar and spindle-associated protein 1 (NUSAP1), and kinesin family member 23 (KIF23), could be instrumental in the development, invasion, or recurrence of glioblastoma. The identification of DEGs and key genes in this study advances our comprehension of the molecular pathways that contribute to the oncogenesis and progression of glioblastoma. This research provides valuable insights into potential diagnostic and therapeutic targets for glioblastoma.

Downregulation of CIAPIN1 regulates the proliferation, migration and glycolysis of breast cancer cells via inhibition of STAT3 pathway

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a protein that regulates apoptosis and programmed cell death. This research aims to evaluate its potential role in inhibiting breast cancer cell proliferation, migration, and glycolysis and uncover its underlying molecular mechanism. We collected breast cancer tissue samples from eight patients between January 2019 and June 2023 in our Hospital to analyse CIAPIN1 expression. We transfected human breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-453, and MDA-MB-468) with siRNA of CIAPIN1. Finally, we determined protein expression using RT-qPCR and Western blotting. CIAPIN1 expression was elevated in both breast cancer tissue and serum. Overexpression of CIAPIN1 detected in the breast cancer cell lines MCF7 and MDA-MB-468. In addition, CIAPIN1 overexpression increased cell proliferation and migration rate. CIAPIN1 downregulation suppressed cell proliferation while elevated cellular apoptosis, reactive oxygen species (ROS) production and oxidative stress in breast cancer cells. Moreover, CIAPIN1 inhibition remarkably suppressed pyruvate, lactate and adenosine triphosphate (ATP) production and reduced the pyruvate kinase M2 (PKM2) protein expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) in breast cancer cells. Downregulation of CIAPIN1 suppresses cell proliferation, migration and glycolysis capacity in breast cancer cells by inhibiting the STAT3/PKM2 pathway.

Antiviral Activity of Angelica Tenuissima Nakai against Influenza A Virus.

The influenza A virus poses a serious threat to human health and is an important global public health issue. The drugs currently used for treatment are becoming increasingly ineffective against influenza A viruses and require the development of new antiviral drugs. Angelica tenuissima Nakai (ATN), a traditional herbal medicine belonging to the Umbelliferae family, exhibits a broad range of pharmacological activities, including inflammation, headache, and cold symptoms. In the present study, based on target protein identification, functional enrichment analysis, and gene set comparisons, we first suggested that ATN has potential therapeutic effects against influenza A virus infection. Next, methylthiazol tetrazolium (MTT) and sulforhodamine B colorimetric (SRB) assay results revealed that ATN exhibited low cytotoxicity in Madin-Darby canine kidney (MDCK) cells. The antiviral properties of ATN were observed against H1N1 and H3N2 virus strains. Microscopy confirmed the increased survival rate of the host cells. Further time-of-addition experiments revealed that the addition of ATN before virus adsorption showed similar results to the whole period of treatment. The pre- and co-treated groups showed lower levels of viral RNA (M1 protein). The results of this study suggest that ATN exhibits antiviral properties against the influenza A virus. These therapeutic properties of ATN can serve as a theoretical basis for further research on the applicability of ATN in the development of antiviral agents.

Novel Abietane type Sugar Triazole Hybrids and Amides against SARS‐CoV‐2 Spike Glycoprotein and Influenza A Virus

Abietane type diterpenic (dehydroabietic, 2,3‐dihydroquinopimaric and maleopimaric) acids were converted by the acid chloride method into a series of aliphatic and heterocyclic amine spacered conjugates. A number of structurally novel derivatives holding 1,2,3‐triazole moieties were designed and synthesized by treating of the propargylated amides and esters with a sugar azides using the Cu(I)‐catalyzed click chemistry approach. The synthesized N‐containing diterpene derivatives were tested for their potential inhibition of influenza A/PuertoRico/8/34 (H1N1) virus in MDCK cell culture and SARS‐CoV‐2 pseudovirus in BHK‐21‐hACE2 cells. Among tested forty‐five compounds ten derivatives were the most efficacious against influenza virus A with IC50 0.7‐63.4 μM together with high selectivity index SI value from 11 from 94. Dihydroquinopimaric acid N‐ethylpiperazine‐amide and dehydroabietic acid 1,2,3‐triazoles spacered with glucose and lactose showed anti‐SARS‐CoV‐2 pseudovirus activity with EC50 values of 1.79‐25.46 µM. Molecular docking and dynamics modeling investigated the binding mode of the lead compounds into the binding pocket of influenza A virus M2 protein and the RBD domain of SARS‐CoV‐2 spike glycoprotein.

Incompatible packaging signals and impaired protein functions hinder reassortment of bat H17N10 or H18N11 segment 7 with human H1N1 influenza A viruses.

Reassortment is one of the mechanisms in fast evolution of influenza A viruses (IAVs) and responsible for generating pandemic strains. To date, why novel bat IAVs are incapable of reassorting with conventional IAVs remains completely understood. Here, we attempted to rescue recombinant PR8 viruses with M segment from bat IAVs to understand the molecular mechanisms in hindering their reassortment. Results showed that bat influenza NEP and M1 have similar functions as respective counterparts of PR8 to medicating viral ribonucleoprotein nuclear export. Moreover, the incompatible packaging signals of M genes from bat and conventional IAVs and impaired bat M2 functions are the major reasons to hinder their reassortment. Recombinant PR8 viruses with bat influenza M open reading frames were generated but showed attenuation, which correlated with the functions of the bat M2 protein. Our studies provide novel insights into the molecular mechanisms that restrict reassortment between bat and conventional IAVs.

Improved cellular immune response induced by intranasal boost immunization with chitosan coated DNA vaccine against H9N2 influenza virus challenge

The H9N2 avian influenza virus (AIV) is spreading worldwide. Presence of H9N2 virus tends to increase the chances of infection with other pathogens which can lead to more serious economic losses. In a previous study, a regulated delayed lysis Salmonella vector was used to deliver a DNA vaccine named pYL233 encoding M1 protein, mosaic HA protein and chicken GM-CSF adjuvant. To further increase its efficiency, chitosan as a natural adjuvant was applied in this study. The purified plasmid pYL233 was coated with chitosan to form a DNA containing nanoparticles (named CS233) by ionic gel method and immunized by intranasal boost immunization in birds primed by oral administration with Salmonella strain. The CS233 DNA nanoparticle has a particle size of about 150 nm, with an encapsulation efficiency of 93.2 ± 0.12 % which protected the DNA plasmid from DNase I digestion and could be stable for a period of time at 37°. After intranasal boost immunization, the CS233 immunized chickens elicited higher antibody response, elevated CD4+ T cells and CD8+ T cells activation and increased T-lymphocyte proliferation, as well as increased productions of IL-4 and IFN-γ. After challenge, chickens immunized with CS233 resulted in the lowest levels of pulmonary virus titer and viral shedding as compared to the other challenge groups. The results showed that the combination of intranasal immunization with chitosan-coated DNA vaccine and oral immunization with regulatory delayed lytic Salmonella strain could enhance the immune response and able to provide protection against H9N2 challenge.

The M2 Protein of the Influenza A Virus Interacts with PEX19 to Facilitate Virus Replication by Disrupting the Function of Peroxisome.

The peroxisomal biogenesis factor 19 (PEX19) is necessary for early peroxisomal biogenesis. PEX19 has been implicated in the replication of a variety of viruses, but the details pertaining to the mechanisms of how PEX19 engages in the life cycle of these viruses still need to be elucidated. Here, we demonstrated that the C terminus of PEX19 interacted with the cytoplasmic tail region of the M2 protein of the influenza A virus (IAV) and inhibited the viral growth titers. IAV infection or PEX19 knockdown triggered a reduction in the peroxisome pool and led to the accumulation of ROS and cell damage, thereby creating favorable conditions for IAV replication. Moreover, a reduction in the peroxisome pool led to the attenuation of early antiviral response mediated by peroxisome MAVS and downstream type III interferons. This study also showed that the interaction between IAV M2 and PEX19 affected the binding of PEX19 to the peroxisome-associated protein PEX14 and peroxisome membrane protein 24 (PMP24). Collectively, our data demonstrate that host factor PEX19 suppresses the replication of the IAV, and the IAV employs its M2 protein to mitigate the restricting role of PEX19.