Growth Of Influenza Virus Research Articles

The Screening and Mechanism of Influenza-Virus Sensitive MDCK Cell Lines for Influenza Vaccine Production.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin-Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents

We previously reported that bakuchiol, a phenolic isoprenoid anti-cancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney (MDCK) cell lysates and purified the specific bakuchiol-binding proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using nano-liquid chromatography with tandem mass spectrometry analysis, we identified prohibitin (PHB) 2, voltage dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and co-localized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein (NP) expression levels and viral titers in the conditioned medium of virus-infected MDCK cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Generation of a Porcine Cell Line Stably Expressing Pig TMPRSS2 for Efficient Isolation of Swine Influenza Virus.

Pigs are important animals for meat production but can carry several zoonotic diseases, including the Japanese encephalitis virus, Nipah virus, and influenza viruses. Several Orthomyxoviridae and Coronavirinae respiratory viruses require cleavage of envelope proteins to acquire viral infectivity and consequently, need a host protease or the addition of exogenous trypsin for efficient propagation. Host TMPRSS2 is a key protease responsible for viral cleavage. Stable expression of human TMPRSS2 in African green monkey-derived Vero cells can enhance the porcine epidemic diarrhea virus. However, considering the narrow host tropism of viruses, a porcine cell line expressing pig TMPRSS2 could be optimal for replicating pig-derived viruses. Herein, we generated and evaluated a pig-derived PK-15 cell line stably expressing pig TMPRSS2. This cell line markedly (>1000-fold) and specifically enhanced the growth of influenza viruses. Furthermore, we demonstrated the usefulness of a PK-15 cell line lacking the Stat2 gene with a stable expression of pig TMPRSS2 for efficient virus isolation from clinical samples in the presence of type I interferons. Therefore, PK-15 cells expressing pig TMPRSS2 could be a valuable and promising tool for virus isolation, vaccine production, and virological studies of TMPRSS2-dependent viruses.

Antiviral Activity of Marine Actinomycetes, Saccharopolyspora jiangxiensis IMA1 against Influenza A/(H1N1) pdm09

Influenza viruses are major communicable pathogens responsible for respiratory diseases affecting millions worldwide and denoted by increased morbidity and significant mortality. Antiviral drugs and periodical vaccination are used to control Influenza infections. The utility of currently available drugs is of major concern due to emergence of drug resistance. This necessitates the development of novel antiviral drugs from natural resources. Broad arsenal of highly effective novel anti-influenza drugs can be developed from actinomycetes which have been explored for development of an array of antimicrobials. Fractions of methanol, ethanol, ethyl acetate and aqueous of the Saccharopolyspora jiangxiensis IMA1 were employed to assess the antiviral activity against Oseltamivir resistant influenza A/(H1N1)pdm09 virus. MTT, Plaque Reduction, Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and DAPI staining assays were performed to validate the study findings. Selectivity Index value of 18.38µg/ml concentration of the fraction was found to be effective to inhibit the growth of influenza viruses employing the Madin-Darby Canine Kidney cell line. Fraction produced a visually noticeable reduction in cytopathic effect as well as a reduction in viral titre as determined by the reduction in plaque formation. qRT-PCR assay clearly showed a linear relationship between the fraction concentration and the Ct values, demonstrating the virus growth inhibitory activity of the fraction. S.jiangxiensis IMA1 ethyl acetate fraction showed promising antiviral activity as revealed by inhibiting the amplification of influenza virus type A/(H1N1)pdm09. The research findings will be useful for the development of new antiviral drug from the bioactive actinomycetes extractions.

Development of an Enhanced High-Yield Influenza Vaccine Backbone in Embryonated Chicken Eggs.

Vaccination is an efficient approach to preventing influenza virus infections. Recently, we developed influenza A and B virus vaccine backbones that increased the yield of several vaccine viruses in Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cells. These vaccine backbones also increased viral replication in embryonated chicken eggs, which are the most frequently used platform for influenza vaccine manufacturing. In this study, to further increase the viral titers in embryonated chicken eggs, we introduced random mutations into the 'internal genes' (i.e., all influenza viral genes except those encoding the hemagglutinin and neuraminidase proteins) of the influenza A virus high-yield virus backbone we developed previously. The randomly mutated viruses were sequentially passaged in embryonated chicken eggs to select variants with increased replicative ability. We identified a candidate that conferred higher influenza virus growth than the high-yield parental virus backbone. Although the observed increases in virus growth may be considered small, they are highly relevant for vaccine manufacturers.

Autophagy Markers Induced by Influenza Virus and MUC1 Expressions in Cancer-Derived Cell Lines

Influenza virus-induced autophagy is often accompanied by apoptosis and results in cell death in virus-infected cells. It is well known that autophagy is modulated by the mTOR/PI3K/Akt pathway, which plays an important role in the response to the presence of energy sources and external stimuli. This pathway is modulated by mucin 1 (MUC1), which has extracellular and intracellular components and plays an important role in metastasis and chemotherapeutic resistance. In this study, it was aimed to investigate the expression of MUC1 after the inoculation of influenza viruses into the cancer-derived cell cultures and, accordingly, the changes in autophagy markers such as mTOR and LC3B. In this study, MCF-7, HeLa and A-549 cell lines were used which have adenocarcinoma origin. To control the growth of influenza virus in these cells, the MDCK cell line was also inoculated. Centrifuge-enhanced shell-vial cell culture method was used in all experiments. Influenza A (H1N1) pdm09 strain was inoculated into these cell lines then the expressions of viral nucleic acid and cycle threshold (Ct) of MUC1, mTOR, LC3B associated genes were investigated by quantitative real-time reverse transcriptase polymerase chain reaction (qRTPCR) method in the samples taken from the supernatants of all cells at the end of the 48-hour incubation period. To investigate whether these markers were present in cells, after all cells were permeabilized with paraformaldehyde, cell-coated infected coverslips were stained with fluorescent labeled monoclonal antibodies developed against MUC1, mTOR, LC3B and influenza virus antigens. In the examination of fluorescence microscopy, all of the cell cultures (MCF-7, He-La and A-549) infected with influenza virus yielded positive results in terms of LC3B, mTOR and MUC1 monoclonal antibody staining, whereas all of the non-infected cells were found negative. Cycle threshold values of MUC1, LC3B and mTOR associated genes were found to be lower in A-549 cell line inoculated with influenza virus. Although protein expression was demonstrated in MCF-7 and He-La cell lines, similar changes were not detected in the 1/Ct values of genes in the autophagy pathway. The Ct value of the MUC1 gene was found to be higher only in the MCF-7 cell line after inoculation. In conclusion, it was observed that the specific expression pattern for influenza virus-induced autophagy was formed only in the A-549 cell line among the adenocarcinoma cells. It was thought that this relationship could constitute a dataset in further research on lung adenocarcinoma. However, in future studies, the determination of the expression of these genes at the protein level by using further tests will provide better comparison of the results.

Impact of Baloxavir Resistance-Associated Substitutions on Influenza Virus Growth and Drug Susceptibility.

Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. IMPORTANCE Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.

Key Amino Acids of M1-41 and M2-27 Determine Growth and Pathogenicity of Chimeric H17 Bat Influenza Virus in Cells and in Mice.

Based on our previous studies, we show that the M gene is critical for the replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing the bat M gene with those from human and swine influenza A viruses. However, the key amino acids of the M1 and/or M2 proteins that are responsible for virus replication and pathogenicity remain unknown. In this study, replacement of the PR8 M gene with the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of the chimeric H17 bat influenza virus. Further studies revealed that M1 was more crucial for viral growth and pathogenicity than M2 and that the amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins identified in this study might be important for influenza virus surveillance and could be used to produce live attenuated vaccines in the future. IMPORTANCE The M1 and M2 proteins influence the morphology, replication, virulence, and transmissibility of influenza viruses. Although a few key residues in the M1 and M2 proteins have been identified, whether other residues of the M1 and M2 proteins are involved in viral replication and pathogenicity remains to be discovered. In the background of the chimeric H17 bat influenza virus, the Eurasian avian-like M gene from the A/California/04/2009 virus significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 in viral growth and pathogenicity in vitro and in vivo and that the key amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine applications in the future. These findings provide important contributions to knowledge of the genetic basis of the virulence of influenza viruses.

CACO-2 cells: A continuous cell line with sensitive and broad-spectrum utility for respiratory virus culture

BackgroundPrimary rhesus monkey kidney cells (RhMK) can be used for the detection of respiratory viruses, including influenza and parainfluenza. The human colon adeno-carcinoma cell line, CACO-2, has been previously used for the growth of multiple influenza viruses, including seasonal, novel and avian lineages. ObjectiveWe compared CACO-2, Madin-Darby Canine Kidney (MDCK), and RhMK cells for the isolation of viruses from patients presenting with influenza like-illness (ILI). Study designNasopharyngeal specimens from patients with ILI in primary care settings were processed for conventional viral culture in MDCK, RhMK, and CACO-2. Cells were examined microscopically for cytopathic effect (CPE) and confirmatory testing included immunofluorescent antigen (IFA) detection and real-time RT-PCR. Additionally, 16 specimens positive for respiratory syncytial virus (RSV) by PCR were inoculated on CACO-2 cells. Statistical analysis was done using Chi-square test with IBM Statistical Program. ResultsOf 1031 respiratory specimens inoculated, viruses were isolated and confirmed from 331 (32.1 %) in MDCK cells, 304 (29.5 %) in RhMk cells, and 433 (42.0 %) in CACO-2 cells. These included influenza A/(H1N1)pdm09, influenza A(H3N2), influenza B, parainfluenza virus (PIV) types 1, 2, and 3, human coronavirus 229E (CoV-229E), human adenovirus (HAdV), herpes simplex virus 1 (HSV 1), and enterovirus (EV). Influenza A viruses grew best in the CACO-2 cell line. Time to observation of CPE was similar for all three cell types but unlike RhMK and MDCK cells, virus-specific morphological changes were indistinguishable in CACO-2 cells. None of the 16 specimens positive for RSV by PCR grew on CACO-2 cells. ConclusionsThe overall respiratory virus culture isolation rate in CACO-2 cells was significantly higher than that in RhMK or MDCK cells (p < 0.05). CACO-2 cells also supported the growth of some viruses that did not grow in either RhMK or MDCK cells. Except for RSV, CACO-2 cells provide a worthwhile addition to culture algorithms for respiratory specimens.

A humanized MDCK cell line for the efficient isolation and propagation of human influenza viruses.

Here, we developed hCK, a Madin-Darby canine kidney (MDCK) cell line that expresses high levels of human influenza virus receptors and low levels of avian virus receptors. hCK cells supported human A/H3N2 influenza virus isolation and growth much more effectively than conventional MDCK or human virus receptor-overexpressing (AX4) cells. A/H3N2 viruses propagated in hCK cells also maintained higher genetic stability than those propagated in MDCK and AX4 cells.

Inhibition of Influenza Virus Infection by Lentinus edodes Mycelia Extract Through Its Direct Action and Immunopotentiating Activity.

Lentinula edodes mycelia (LEM) solid culture extracts contain many bioactive compounds with diverse pharmacological activities such as antitumor, antiviral, and immunopotentiating effects. In this study, we examined the anti-influenza virus activity of LEM in vitro and in vivo. LEM directly inhibited influenza virus growth in vitro at early phases of infection, possibly at the entry process of viral particles to host cells. We also found that the nasal administration of LEM increased the survival rate of infected mice, and this was likely due to the direct action of LEM on the viral growth. The oral administration of LEM showed prolonged median survival time of infected mice. Histological analysis revealed that the moderate bronchiolitis was observed in infected mice by the oral administration with LEM, and the extent of alveolitis was dramatically reduced. The orally LEM-administered mice showed a rapid activation of IFN-β gene expression upon influenza virus infection. These results suggest that the immunopotentiation activity of LEM on type I IFN pathway represses the virus spread to distal alveolar regions from peribronchiolar regions which are primary infection sites in the mouse model. We propose that LEM has anti-influenza virus activities through the direct action on viral growth and stimulatory activity of innate immunity.

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.

Influenza B virus reverse genetic backbones with improved growth properties in the EB66® cell line as basis for vaccine seed virus generation

Vaccination remains the best available prophylaxis to prevent influenza virus infections, yet current inadequacies in influenza virus vaccine manufacturing often lead to vaccine shortages at times when the vaccine is most needed, as it was the case during the last influenza virus pandemic. Novel influenza virus vaccine production systems will be crucial to improve public health and safety. Here we report the optimization of influenza B virus growth in the proprietary EB66® cell line, currently in use for human vaccine production. To this end, we collected, curated and sequenced 71 influenza B viruses selected for high diversity in date of isolation and lineage. This viral collection was tested for ability to enter and replicate within EB66® cells in a single cycle assay and appears to readily infect these cells. When the collection was tested for viral progeny production in a multi-cycle assay, we found a large variation from strain to strain. The strains with the top growth characteristics from the B/Victoria and B/Yamagata lineages were selected for vaccine backbone generation using a reverse genetics system. We then showed that these backbones maintain their desirable growth within EB66® cells when the HA and NA from poorly growing strains were substituted for the parental segments, indicating that the selected backbones are viable options for vaccine production in EB66®. Finally, we show that compounds previously reported to enhance influenza virus growth in cell culture also increase virus production in the EB66® cell line.

The first external quality assessment of isolation and identification of influenza viruses in cell culture in the Asia Pacific region, 2016

BackgroundThe isolation and propagation of influenza viruses from clinical specimens are essential tools for comprehensive virologic surveillance. Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays. ObjectivesTo conduct an external quality assessment (EQA) of proficiency for isolation and identification of influenza viruses using cell culture techniques among National Influenza Centres (NICs) in the World Health Organisation (WHO) South East Asia and Western Pacific Regions. Study designTwenty-one NICs performed routine influenza virus isolation and identification techniques on a proficiency testing panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to determine their capacity to measure hemagglutination titer and the other 15 samples were used for virus isolation and identification. ResultsAll NICs performed influenza virus isolation using Madin Darby canine kidney (MDCK) or MDCK-SIAT-1 cells. If virus growth was detected, the type, subtype and/or lineage of virus present in isolates was determined using immunofluorescence, RT-PCR and/or hemagglutination inhibition (HI) assays. Most participating laboratories could detect influenza virus growth and could identify virus amplified from EQA samples. However, some laboratories failed to isolate and identify viruses from EQA samples that contained lower titres of virus, highlighting issues regarding the sensitivity of influenza virus isolation methods between laboratories. ConclusionThis first round of EQA was successfully conducted by NICs in the Asia Pacific Region, revealing good proficiency in influenza virus isolation and identification.

Influenza vaccines manufacturing in continuous cell lines: problems and solutions

ABSTRACT In order to decrease the morbidity and mortality caused by seasonal influenza outbreaks, several hundred million vaccine doses are produced worldwide each year. The predominant substrate for the production of the influenza vaccine today is fertilized hen’s eggs. The substitution of the technology based on living organisms by the cell culture-based process offers many advantages, including easier scalability and reduced dependence on the availability of eggs. The African green monkey kidney and Madin Darby canine kidney cell lines support the efficient growth of influenza viruses of different subtypes and, therefore, are considered to be the two most promising alternative substrates for the production of the human influenza vaccine. However, the pH of endosomes in both of these cell lines is higher than the pH essential for triggering a conformational change of the hemagglutinin (HA) of human influenza viruses, which enables the viral-cellular membrane fusion. This mismatch gives rise to mutations in the HA that lead to an increase of the optimum pH of HA conformational change. As of a result of these mismatches, the HA, and consequently the whole virus, has reduced stability to low pH and elevated temperatures. The production of a vaccine from less stable virus will lead to an elevated HA content in the low pH conformation that can affect the safety, potency, infectivity, and protective efficacy of the final inactivated and live attenuated influenza vaccines. The main limitations of the cell line-based influenza vaccine technology and the possibilities to preserve the viral stability over the course of influenza vaccine production are discussed in the review.

Amino acid changes in PB2 and HA affect the growth of a recombinant influenza virus expressing a fluorescent reporter protein.

Influenza viruses that express reporter proteins are useful tools, but are often attenuated. Recently, we found that an influenza virus encoding the Venus fluorescent protein acquired two mutations in its PB2 and HA proteins upon mouse adaptation. Here, we demonstrate that the enhanced viral replication and virulence in mice of this Venus-expressing influenza virus are primarily conferred by the PB2-E712D mutation, with only a minor contribution by the HA-T380A mutation.

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding themechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.

Protective effects of Yinhuapinggan granule on mice with influenza viral pneumonia

Yinhuapinggan granule (YHPG), a Chinese medicine granule on the basis of Ma-Huang-Tang (Ephedra Decoction) and the clinical experience of Professor Wan Haitong, has been shown to inhibit the growth of influenza virus in vitro. The aim of this study was to investigate the protective effects of YHPG on mice with influenza viral pneumonia and its effects on regulating related inflammatory cytokines in influenza virus A-infected mice. ICR mice were inoculated intranasally with 15 LD50 viral dose of influenza virus A/PR/8/34 (H1N1) and treatments with YHPG (7.5, 15 and 30g/kg) were orally administrated daily for 5 consecutive days after challenge, respectively. The results showed that mortality rate, lung index, lung histopathological changes, IL-6 and TNF-α in serum were significantly attenuated in the treatment of YHPG (15 and 30g/kg) than those in the IFV control group, while the levels of IL-2 was significantly enhanced. Moreover, the RT-PCR results revealed that YHPG (15 and 30g/kg) significantly depressed the expressions of IL-1β, IFN-γ and TNF-α mRNA in lung tissues. Furthermore, the immunohistochemical staining results also revealed that the expression of NF-κB p65 proteins was downregulated when treated with YHPG (15 and 30g/kg). These results showed YHPG has protective effects on IFV-infected mice, due to its ability of alleviation of lung damage, regulation of the cytokine production via inhibiting the NF-κB p65 activation, attenuation of systemic and pulmonary inflammatory responses.

In Vitro Virucidal Effect of Mouthrinse Containing C31G on Seasonal Influenza Viruses

C31G is a potent antimicrobial agent and can disrupt the microbial membrane by the alkyl portion of the molecule. The objective of this study was to evaluate the virucidal effectiveness of C31G and mouthrinse containing C31G (Sense-Time) on seasonal influenza viruses. Evaluation of the virucidal activity against influenza viruses was performed with end-point titration in 10-day-old chicken embryos and Madin-Darby canine kidney cells. In vitro studies demonstrated that C31G and Sense-Time inhibited the growth of seasonal influenza viruses even in the presence of 5% organic material. Gargling with C31G or Sense-Time would enhance oropharyngeal hygiene, which would be helpful for reducing influenza transmission.

Proteasomal Serine Hydrolases Are Up-Regulated by and Required for Influenza Virus Infection

Interactions between viruses and their host cells are important determinants of virus replication and of immune responses to the virus. However, these interactions and resulting consequences of these interactions remain poorly defined. Numerous recent quantitative proteomic approaches have measured host proteins affected by virus infection. Here, we used activity-based protein profiling (ABPP) to measure functional alterations in host serine hydrolases after influenza A virus infection of Madin-Darby canine kidney and human A549 lung cells. We identified 62 serine proteases. We then combined the ABPP approach with stable isotope labeling to directly measure how serine hydrolase activities were affected by virus infection. Differentially regulated SHs mapped into a few key cellular pathway systems, most notably the proteasomal system. The specific serine protease inhibitors Aprotinin and Pefablock and specific proteasomal inhibitors Bortezomib and MG132 significantly inhibited influenza virus growth. Some inhibitors also down-regulated activities of several proteasomal proteins, including PSMA1, PSMA2, and PMSB3. Genetic knockdown of PMSA2 also attenuated influenza virus replication. These findings further our understanding of enzymatic cellular processes affected by influenza virus and may be beneficial in the search for additional antiviral therapeutic targets.