Unveiling the role of host kinases at different steps of influenza A virus life cycle.

Influenza viruses remain a major public health concern causing contagious respiratory illnesses that result in around 290,000-650,000 global deaths every year. Their ability to constantly evolve through antigenic shifts and drifts leads to the emergence of newer strains and resistance to existing drugs and vaccines. To combat this, there is a critical need for novel antiviral drugs through the introduction of host-targeted therapeutics. Influenza viruses encode only 14 gene products that get extensively modified through phosphorylation by a diverse array of host kinases. Reversible phosphorylation at serine, threonine, or tyrosine residues dynamically regulates the structure, function, and subcellular localization of viral proteins at different stages of their life cycle. In addition, kinases influence a plethora of signaling pathways that also regulate virus propagation by modulating the host cell environment thus establishing a critical virus-host relationship that is indispensable for executing successful infection. This dependence on host kinases opens up exciting possibilities for developing kinase inhibitors as next-generation anti-influenza therapy. To fully capitalize on this potential, extensive mapping of the influenza virus-host kinase interaction network is essential. The key focus of this review is to outline the molecular mechanisms by which host kinases regulate different steps of the influenza A virus life cycle, starting from attachment-entry to assembly-budding. By assessing the contributions of different host kinases and their specific phosphorylation events during the virus life cycle, we aim to develop a holistic overview of the virus-host kinase interaction network that may shed light on potential targets for novel antiviral interventions.

Twenty natural amino acid substitution screening at the last residue 121 of influenza A virus NS2 protein reveals the critical role of NS2 in promoting virus genome replication by coordinating with viral polymerase.

Both influenza A virus genome transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the influenza RNA polymerase (FluPol), are dynamically regulated across the virus life cycle. It has been reported that the last amino acid I121 of the viral NS2 protein plays a critical role in promoting viral genome replication in influenza mini-replicon systems. Here, we performed a 20 natural amino acid substitution screening at residue NS2-I121 in the context of virus infection. We found that the hydrophobicity of the residue 121 is essential for virus survival. Interestingly, through serial passage of the rescued mutant viruses, we further identified adaptive mutations PA-K19E and PB1-S713N on FluPol which could effectively compensate for the replication-promoting defect caused by NS2-I121 mutation in the both mini-replicon and virus infection systems. Structural analysis of different functional states of FluPol indicates that PA-K19E and PB1-S713N could stabilize the replicase conformation of FluPol. By using a cell-based NanoBiT complementary reporter assay, we further demonstrate that both wild-type NS2 and PA-K19E/PB1-S713N could enhance FluPol dimerization, which is necessary for genome replication. These results reveal the critical role NS2 plays in promoting viral genome replication by coordinating with FluPol.IMPORTANCEThe intrinsic mechanisms of influenza RNA polymerase (FluPol) in catalyzing viral genome transcription and replication have been largely resolved. However, the mechanisms of how transcription and replication are dynamically regulated remain elusive. We recently reported that the last amino acid of the viral NS2 protein plays a critical role in promoting viral genome replication in an influenza mini-replicon system. Here, we conducted a 20 amino acid substitution screening at the last residue 121 in virus rescue and serial passage. Our results demonstrate that the replication-promoting function of NS2 is important for virus survival and efficient multiplication. We further show evidence that NS2 and NS2-I121 adaptive mutations PA-K19E/PB1-S713N regulate virus genome replication by promoting FluPol dimerization. This work highlights the coordination between NS2 and FluPol in fulfilling efficient genome replication. It further advances our understanding of the regulation of viral RNA synthesis for influenza A virus.

CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Chemical intervention of influenza virus mRNA nuclear export.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.

Influenza and antiviral resistance: an overview.

Influenza affects approximately 1 billion individuals each year resulting in between 290,000 and 650,000 deaths. Young children and immunocompromised individuals are at a particularly high risk of severe illness attributable to influenza and these are also the groups of individuals in which reduced susceptibility to neuraminidase inhibitors is most frequently seen. High levels of resistance emerged with previous adamantane therapy for influenza A and despite no longer being used to treat influenza and therefore lack of selection pressure, high levels of adamantane resistance continue to persist in currently circulating influenza A strains. Resistance to neuraminidase inhibitors has remained at low levels to date and the majority of resistance is seen in influenza A H1N1 pdm09 infected immunocompromised individuals receiving oseltamivir but is also seen less frequently with influenza A H3N2 and B. Rarely, resistance is also seen in the immunocompetent. There is evidence to suggest that these resistant strains (particularly H1N1 pdm09) are able to maintain their replicative fitness and transmissibility, although there is no clear evidence that being infected with a resistant strain is associated with a worse clinical outcome. Should neuraminidase inhibitor resistance become more problematic in the future, there are a small number of  alternative novel agents within the anti-influenza armoury with different mechanisms of action to neuraminidase inhibitors and therefore potentially effective against neuraminidase inhibitor resistant strains. Limited data from use of novel agents such as baloxavir marboxil and favipiravir, does however show that resistance variants can also emerge in the presence of these drugs.

A Parallel Phenotypic Versus Target-Based Screening Strategy for RNA-Dependent RNA Polymerase Inhibitors of the Influenza A Virus.

Influenza A virus infections cause significant morbidity and mortality, and novel antivirals are urgently needed. Influenza RNA-dependent RNA polymerase (RdRp) activity has been acknowledged as a promising target for novel antivirals. In this study, a phenotypic versus target-based screening strategy was established to identify the influenza A virus inhibitors targeting the virus RNA transcription/replication steps by sequentially using an RdRp-targeted screen and a replication-competent reporter virus-based approach using the same compounds. To demonstrate the utility of this approach, a pilot screen of a library of 891 compounds derived from natural products was carried out. Quality control analysis indicates that the primary screen was robust for identification of influenza A virus inhibitors targeting RdRp activity. Finally, two hit candidates were identified, and one was validated as a putative RdRp inhibitor. This strategy can greatly reduce the number of false positives and improve the accuracy and efficacy of primary screening, thereby providing a powerful tool for antiviral discovery.

Subclade 2.2.1-Specific Human Monoclonal Antibodies That Recognize an Epitope in Antigenic Site A of Influenza A(H5) Virus HA Detected between 2015 and 2018.

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144-147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144-147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144-147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades.

Eleutheroside B1 mediates its anti-influenza activity through POLR2A and N-glycosylation.

Influenza viruses represent a serious threat to human health. Although our research group has previously demonstrated the antiviral and anti­inflammatory activities of eleutheroside B1, a detailed explanation of the mechanism by which it is effective against the influenza virus remains to be elucidated. In the present study, the transcriptomic responses of influenza A virus­infected lung epithelial cells (A549) treated with eleutheroside B1 were investigated using high­throughput RNA sequencing, and potential targets were identified using a molecular docking technique, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) assay, and DNA methylation analysis. The transcriptomic data revealed that there are 1,871 differentially expressed genes (DEGs) between the cells infected with the influenza virus strain variant PR8, and the cells infected with PR8 and treated with eleutheroside B1. Among the DEGs, RNA polymerase II subunit A (POLR2A; encoding the largest subunit of RNA polymerase II) and mannosidase α class II member 1 (MAN2A1) were selected from the molecular docking analysis with eleutheroside B1. The docking score of Drosophila melanogaster MAN2A1 (3BVT) was 11.3029, whereas that of POLR2A was 9.0133. The RT­qPCR results demonstrated that the expression levels of host genes (MAN2A2, POLR2A) and viral genes (PA, PB1, PB2, HA) were downregulated following eleutheroside B1 treatment. Bisulfite­sequencing PCR was performed to investigate whether eleutheroside B1 was able to modify the DNA methylation of POLR2A, and the results suggested that the average proportion of methylated CpGs (­222­72 bp) increased significantly following treatment with eleutheroside B1. Taken together, these findings suggested that eleutheroside B1 may affect N­glycan biosynthesis, the chemokine signaling pathway, cytokine­cytokine receptor interaction and, in particular, may target the POLR2A to inhibit the production of influenza virus genes.

Phenotypic Lentivirus Screens to Identify Antiviral Single Domain Antibodies.

Our understanding of infection biology is based on experiments in which pathogen or host proteins are perturbed by small compound inhibitors, mutation, or depletion. This approach has been remarkably successful, as, for example, demonstrated by the independent identification of the endosomal membrane protein Niemann-Pick C1 as an essential factor for Ebola virus infection in both small compound and insertional mutagenesis screens (Côté, Nature 477:344-348, 2011; Carette et al., Nature 477:340-343, 2011). However, many aspects of host-pathogen interactions are poorly understood because we cannot target all of the involved molecules with small molecules, or because we cannot deplete essential proteins. Single domain antibody fragments expressed in the cytosol or other organelles constitute a versatile alternative to perturb the function of any given protein by masking protein-protein interaction interfaces, by stabilizing distinct conformations, or by directly interfering with enzymatic activities. The variable domains of heavy chain-only antibodies (VHHs) from camelid species can be cloned from blood samples of animals immunized with the desired target molecules. We can thus exploit the ability of the camelid immune system to generate affinity-matured single domain antibody fragments to obtain highly specific tools. Interesting VHH candidates are typically identified based on their affinity toward immobilized antigens using techniques such as phage display.The phenotypical screening approach described here allows the direct identification of VHHs that prevent infection of cells with influenza A virus (IAV) or other pathogens. The VHH repertoire is cloned into a lentiviral vector, which is used to generate pseudo-typed lentivirus particles. Target cells are transduced with the lentivirus, so that every cell inducibly expresses a different VHH. This cell collection is then challenged with a lethal dose of virus. Only the cells which express a VHH that prevents infection by targeting virus proteins or host cell components essential for infection will survive. We can thus identify critical target molecules including vulnerable epitopes and conformations, render target molecules accessible to informative perturbation studies, and stabilize intermediates of virus entry for detailed analysis.

Immunomodulatory properties of quercetin-3-O-α-L-rhamnopyranoside from Rapanea melanophloeos against influenza a virus.

BACKGROUND: Influenza infection is a major public health threat. The role of influenza A virus-induced inflammatory response in severe cases of this disease is widely recognized. Drug resistance and side effects of chemical treatments have been observed, resulting in increased interest in alternative use of herbal medications for prophylaxis against this infection. The South African medicinal plant, Rapanea melanophloeos (RM) (L.) Mez of the family Myrsinaceae was selected owing to its traditional use for the treatment of several diseases such as respiratory ailments and also previous preliminary studies of anti-influenza activity of its methanolic extract. The aim of this study was to investigate the immunomodulatory properties of a glycoside flavone isolated from RM against influenza A virus. METHODS: The non-cytotoxic concentration of the quercetin-3-O-α-L-rhamnopyranoside (Q3R) was determined by MTT assay and tested for activity against influenza A virus (IAV) in simultaneous, pre-penetration and post-penetration combination treatments over 1 h incubation on MDCK cells. The virus titer and viral load targeting NP and M2 viral genes were determined using HA and qPCR, respectively. TNF-α and IL-27 as pro- and anti-inflammatory cytokines were measured at RNA and protein levels by qPCR and ELISA, respectively. RESULTS: Quercetin-3-O-α-L-rhamnopyranoside at 150 µg/ml decreased the viral titer by 6 logs (p < 0.01) in the simultaneous procedure. The NP and M2 genes copy numbers as viral target genes, calculated based on the Ct values and standard formula, significantly decreased in simultaneous treatment (p < 0.01). The expression of cytokines was also considerably affected by the compound treatment. CONCLUSIONS: This is the first report of quercetin-3-O-α-L-rhamnopyranoside from RM and its immunomodulatory properties against influenza A virus. Further research will focus on detecting the specific mechanism of virus-host interactions.

A Simple and Robust Approach for Evaluation of Antivirals Using a Recombinant Influenza Virus Expressing Gaussia Luciferase.

Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. Because the effectiveness of seasonal vaccines is highly variable and the currently available drugs are limited in their efficacy because of the emergence of drug resistance, there is an urgent need to develop novel antivirals. In this study, we characterized a recombinant IAV-carrying Gaussia luciferase (Gluc) gene and determined its potential as a tool for evaluating therapeutics. We demonstrated that this recombinant IAV is replication-competent in tissue culture and pathogenic in mice, although it is slightly attenuated compared to the parental virus. Luciferase expression correlated well with virus propagation both in vitro and in vivo, providing a simple measure for viral replication in tissue culture and in mouse lungs. To demonstrate the utility of this virus, ribavirin and oseltamivir phosphate were used to treat the IAV-infected cells and mice, and we observed the dose-dependent inhibition of viral replication by a luciferase assay. Moreover, the decreased luciferase expression in the infected lungs could predict the protective efficacy of antiviral interventions as early as day 2 post virus challenge. In summary, this study provides a new and quantitative approach to evaluate antivirals against IAV.

Functional growth inhibition of influenza A and B viruses by liquid and powder components of leaves from the subtropical plant Melia azedarach L.

We evaluated the anti-influenza-virus effects of Melia components and discuss the utility of these components. The effects of leaf components of Melia azedarach L. on viruses were examined, and plaque inhibition tests were performed. The in vivo efficacy of M. azedarach L. was tested in a mouse model. Leaf components of Melia azedarach L. markedly inhibited the growth of various influenza viruses. In an initial screening, multiplication and haemagglutination (HA) activities of H1N1, H3N2, H5, and B influenza viruses were inactivated by the liquid extract of leaves of M. azedarach L. (MLE). Furthermore, plaque inhibition titres of H1N1, H3N2, and B influenza viruses treated with MLE ranged from 103.7 to 104.2. MLE possessed high plaque-inhibitory activity against pandemic avian H5N1, H7N9, and H9N2 vaccine candidate strains, with a plaque inhibition titre of more than 104.2. Notably, the buoyant density decreased from 1.175 to 1.137 g/cm3, and spikeless particles appeared. We identified four anti-influenza virus substances: pheophorbide b, pheophorbide a, pyropheophorbide a, and pheophytin a. Photomorphogenesis inside the envelope may lead to removal of HA and neuraminidase spikes from viruses. Thus, MLE could efficiently remove floating influenza virus in the air space without toxicity. Consistent with this finding, intranasal administration of MLE in mice significantly decreased the occurrence of pneumonia. Additionally, leaf powder of Melia (MLP) inactivated influenza viruses and viruses in the intestines of chickens. MLE and MLP may have applications as novel, safe biological disinfectants for use in humans and poultry.

Protein disulfide isomerases as potential therapeutic targets for influenza A and B viruses.

Seasonal flu as well as potential pandemic flu outbreaks continuously underscores the importance of the preventive and therapeutic measures against influenza viruses. During screening of natural and synthetic small molecules against influenza A and B virus, we identified juniferdin as a highly effective inhibitor against both viruses in cells. Since juniferdin is known to inhibit protein disulfide isomerases (PDIs), multiple PDI inhibitors were tested against these viruses. Among PDI inhibitors, 16F16, PACMA31, isoquercetin, epigallocatechin-3-gallate or nitazoxanide significantly reduced the replication of influenza A and B viruses in MDCK and A549 cells. Furthermore, siRNAs specific to three PDI family members (PDI1, PDIA3 or PDIA4) also significantly reduced the replication of influenza A and B viruses in cells. These results suggest that PDIs may serve as excellent targets for the development of new anti-influenza drugs.

Inhibition of influenza virus replication by adlay tea.

BACKGROUND: The present study was conducted aiming to examine the antiviral activity of adlay tea and its components against influenza viruses. We further aimed to clarify the mechanism by which these components regulate virus replication. RESULTS: Adlay tea at a concentration suitable for drinking inhibited the multiplication of influenza viruses. Moreover, our results suggest that individual components of the tea had antiviral activities against the influenza A/PR/8/34 virus. Adlay tea inhibited multiplication of the H1N1, H3N2 and B types of influenza virus, including oseltamivir-resistant viruses. In addition, adlay tea inhibited influenza infection during the periods of virus adsorption to the cell and virus replication. Adlay tea did not suppress hemagglutination inhibition or cell fusion, although it slightly inhibited virus binding to Malin Darby canine kidney cells. Furthermore, our findings suggest that the antiviral compounds included in adlay tea were ingredients other than polyphenols and that there were several types of effective compounds in adlay tea inhibiting several steps of viral replication. CONCLUSION: The results of the present study demonstrate that adlay tea had antiviral effects against influenza viruses. Our findings with respect to adlay tea suggest that the polyphenols might have a small influence on its antiviral activity and that other ingredients might have more influence. © 2017 Society of Chemical Industry.

Designing and screening of universal drug from neem (Azadirachta indica) and standard drug chemicals against influenza virus nucleoprotein.

BACKGROUND: Different strains of influenza virus are affecting a large number of people worldwide. Many synthetic antiviral medicines are available for influenza virus in the market. But still there is a need for the development of universal drugs against these strains of influenza virus. METHODS: For this purpose conserved residues within the influenza virus nucleoprotein have been retrieved. The drugs, previously known to have antiviral properties, were screened to identify the best candidate universal drug against Influenza virus strains. Compounds from leaf extracts of neem, were also screened to identify the natural drugs without side effects. RESULT: Molecular docking identified three potential compounds (Nimbaflavone, Rutin, and Hyperoside) having perfect binding with reported conserved residues (ASP302, SER50) of influenza virus nucleoprotein that is involved in the binding of drugs. Further analysis showed Hyperoside as a universal drug against various influenza strains. Some chemical drugs were also evaluated through screening against nucleoprotein. The results showed six drugs (OMS, CBX, LGH, Naproxen, BMS-883559, and BMS-885838) which were interacting with same conserved residues (ASP302, TYR52, SER50, GLY288, SER376, and ARG99) as were found in the case of neem phytochemicals. Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. CONCLUSION: The compound Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. So these compounds have been identified for their potential against influenza strains to be utilized as a universal drug.

Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) Induce broadly protective immune responses against divergent influenza subtypes.

The development of a universal influenza vaccine that provides broad cross protection against existing and unforeseen influenza viruses is a critical challenge. In this study, we constructed and expressed conserved sM2 and HA2 influenza antigens with cholera toxin subunit A1 (CTA1) on the surface of Lactobacillus casei (pgsA-CTA1sM2HA2/L. casei). Oral and nasal administrations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and their isotypes (IgG1 & IgG2a) as well as mucosal IgA. The mucosal administration of pgsA-CTA1sM2HA2/L. casei may also significantly increase the levels of sM2- or HA2-specific cell-mediated immunity because increased release of both IFN-γ and IL-4 was observed. The recombinant pgsA-CTA1sM2HA2/L. casei provided better protection of BALB/c mice against 10 times the 50% mouse lethal doses (MLD50) of homologous A/EM/Korea/W149/06(H5N1) or A/Aquatic bird/Korea/W81/2005 (H5N2) and heterologous A/Puerto Rico/8/34(H1N1), or A/Chicken/Korea/116/2004(H9N2) or A/Philippines/2/08(H3N2) viruses, compared with L. casei harboring sM2HA2 and also the protection was maintained up to seven months after administration. These results indicate that recombinant L. casei expressing the highly conserved sM2, HA2 of influenza and CTA1 as a mucosal adjuvant could be a potential mucosal vaccine candidate or tool to protect against divergent influenza viruses for human and animal.

Targeting Influenza A Virus RNA Promoter.

The emergence of drug-resistant strains of influenza virus makes exploring new classes of inhibitors that target universally conserved viral targets a highly important goal. The influenza A viral genome is made up of eight single-stranded RNA-negative segments. The RNA promoter, consisting of the conserved sequences at the 3' and 5' end of each RNA genomic segment, is universally conserved among influenza A virus strains and in all segments. Previously, we reported on the identification and NMR structure of DPQ (6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) (compound 1) in complex with the RNA promoter. Here, we report on additional screening and SAR studies with compound 1, including ex vivo anti-influenza activity assays, resulted in improved cellular activity against influenza A virus in the micromolar range.

Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses.

Influenza A viruses (IAVs), particularly H1N1, H5N1 and H7N9, pose a substantial threat to public health worldwide. Here, we report that MIR2911, a honeysuckle (HS)-encoded atypical microRNA, directly targets IAVs with a broad spectrum. MIR2911 is highly stable in HS decoction, and continuous drinking or gavage feeding of HS decoction leads to a significant elevation of the MIR2911 level in mouse peripheral blood and lung. Bioinformatics prediction and a luciferase reporter assay showed that MIR2911 could target various IAVs, including H1N1, H5N1 and H7N9. Synthetic MIR2911 significantly inhibited H1N1-encoded PB2 and NS1 protein expression, but did not affect mutants in which the MIR2911-binding nucleotide sequences were altered. Synthetic MIR2911, extracted RNA from HS decoction and HS decoction all significantly inhibited H1N1 viral replication and rescued viral infection-induced mouse weight loss, but did not affect infection with a mutant virus in which the MIR2911-binding nucleotide sequences of PB2 and NS1 were altered. Importantly, the inhibitory effect of HS decoction on viral replication was abolished by an anti-MIR2911 antagomir, indicating that the physiological concentration of MIR2911 in HS decoction could directly and sufficiently suppress H1N1 viral replication. MIR2911 also inhibited H5N1 and H7N9 viral replication in vitro and in vivo. Strikingly, administration of MIR2911 or HS decoction dramatically reduced mouse mortality caused by H5N1 infection. Our results demonstrate that MIR2911 is the first active component identified in Traditional Chinese Medicine to directly target various IAVs and may represent a novel type of natural product that effectively suppresses viral infection.

Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss.

BACKGROUND: Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form. RESULTS: The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach. CONCLUSION: rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.