Influenza Virus Particles Research Articles

Preparation and protection of monoclonal antibody against H7N9 influenza virus hemagglutinin

The first human infection with novel H7N9 influenza virus was reported in March 2013 in China. To date, the H7N9 influenza viruses possess a potential pandemic threat to public health worldwide and have caused severe infection and high mortality in humans in China. The latent period of human infection of H7N9 influenza virus is generally within 7 days. Patients usually show flu like symptoms, such as runny nose, sore throat, fever, cough, headache and muscle pain. Severe patients have rapid development, characterized by severe pneumonia and dyspnea. Antibodies play a major role in protective immunity against the influenza virus infection, and passive immunization with monoclonal antibodies (MAbs) specific to viral proteins might be a potential option for humans against lethal infection of the influenza viruses. It is known that hemagglutinin (HA), is the major viral antigenic protein on the influenza virus particle and the primary target of neutralizing antibodies against influenza virus. In this study, in order to obtain the HA MAbs of influenza virus H7N9 (A/Shanghai/2/2013), study their biology characteristics, and evaluate the prophylactic and therapeutic efficacy of the HA MAbs in mouse model. The BALB/c mice were immunized with inactivated H7N9 vaccine by subcutaneous administration for three times, and boosted with inactivated H7N9 vaccine by the tail vein three days before fusion. Finally, 28 MAbs were obtained and identified by ELISA and hemagglutination inhibition test (HI). Eight IgG subtype MAbs (1A8, 2A12, 2H3, 4A11, 4C8, 4G5, 6A3, 7G2) were selected to identify their biology characteristics. The ELISA titers of ascites of the eight MAbs were 102–106. The eight Mabs exhibited high affinity which can reach nmol/L level and meet the requirement of affinity for monoclonal antibody drug. The immunofluorescence assay indicated that the eight MAbs had excellent specificity for HA protein expressed in 293T cell and were able to recognize the H7N9 influenza virus particles. In addition, all the eight MAbs could inhibit HA activity and the MAbs 2H3, 4A11, 4G5 and 6A3 can also present neutralization activity against the H7N9 live virus. The highest neutralizing titer was up to 1:14125. At the same time, we evaluated the prophylactic and therapeutic efficacy of these MAbs against the homologous H7N9 strain. The activity against H7N9 virus was evaluated by clinical syndromes, survival rates and lung viral titers. The results showed that in the prophylactic and therapeutic experiments, MAb 6A3 can inhibit effectively H7N9 influenza virus infection, provided 100% protection and remarkably reduced the lung viral titers in mouse model. The HA monoclonal antibodies against influenza virus H7N9 were successfully obtained. The HA MAbs not only may be as a prophylactic and therapeutic measure to prevent H7N9 influenza virus infection, but also laid the foundation for us to research the H7N9 HA antigen epitope and develop diagnostic reagents.

Comparison of the Filter Efficiency of Medical Nonwoven Fabrics against Three Different Microbe Aerosols.

Exact evaluation of the performance of surgical masks and biohazard protective clothing materials against pathogens is important because it can provide helpful information that healthcare workers can use to select suitable materials to reduce infection risk. Currently, to evaluate the protective performance of nonwoven fabrics used in surgical masks against viral aerosols, a non-standardized test method using phi-X174 phage aerosols is widely performed because actual respiratory viruses pose an infection risk during testing and the phage is a safe virus to humans. This method of using a phage is simply modified from a standard method for evaluation of filter performance against bacterial aerosols using Staphylococcus aureus, which is larger than virus particles. However, it is necessary to perform such evaluations based on the size of the actual pathogen particles. Thus, we developed a new method that can be performed safely using inactivated viral particles and can quantitate the influenza virus in aerosols by antigen-capture ELISA (Shimasaki et al., 2016a) . In this study, we used three different microbial aerosols of phi-X174 phage, influenza virus, and S. aureus and tested the filter efficiency by capturing microbial aerosols for two medical nonwoven fabrics. We compared the filter efficiency against each airborne microbe to analyze the dependency of filter efficiency on the microbial particle size. Our results showed that against the three types of spherical microbe particles, the filter efficiencies against influenza virus particles were the lowest and those against phi-X174 phages were the highest for both types of nonwoven fabrics. The experimental results mostly corresponded with theoretical calculations. We conclude that the filter efficiency test using the phi-X174 phage aerosol may overestimate the protective performance of nonwoven fabrics with filter structure compared to that against real pathogens such as the influenza virus.

Cellulose Monolith with Tunable Functionality for Immobilization of Influenza Virus

Advancements in influenza virus or virus like particle separation technology can greatly accelerate vaccine production. Due to frequent mutation of the influenza virus, rapid and effective separation of influenza virus directly from host cells still remains a challenge. In this paper, we developed a new cellulose fiber monolith-based chromatography with tunable anion exchange and pseudo-affinity modifications for the separation of cell cultureproduced influenza virus. A novel strategy is proposed for preparing cellulose fiber monolith, which enhances the mechanical property of the monolith. Controllable pore size and porosity were achieved by dispersing and removing a porogen into cellulose-dissolving solution. The structure of cellulose monolith was characterized by Scanning Electron Microscope and the porosity study, which confirmed the controllable average pore size of 10 micron and porosity up to 85%. Two virus separation modes including anion-exchange and pseudo-affinity were introduced into the cellulose monolith, subsequently. The two modifications were quantified by an Element Analyzer. A H1N1 influenza virus strain (A/WSN/33) was produced and harvested in adherent Madin-Darby Canine Kidney Epithelial (MDCK) cells and used directly for virus separation. The results indicated that the pseudo-affinity mode had higher virus separation efficiency and had less host cell dsDNA and protein contamination.

Mutations in the Influenza A Virus M1 Protein Enhance Virus Budding To Complement Lethal Mutations in the M2 Cytoplasmic Tail.

The influenza A virus M1 and M2 proteins play important roles in virus assembly and in the morphology of virus particles. Mutations in the distal cytoplasmic tail region of M2, and in particular a tyrosine-to-alanine mutation at residue 76 (Y76A), were essential for infectious virus production and filament formation while having limited effects on total virus particle budding. Using a novel selection method, mutations at seven different M1 amino acids (residue 73, 94, 135, 136, or 138 or a double mutation, 93/244) that are not found in circulating influenza virus strains or have not been previously identified to play a role in influenza A virus assembly were found to complement the lethal M2Y76A mutation. These M1 suppressor mutations restored infectious virus production in the presence of M2Y76A and mediated increased budding and filament formation even in the absence of M2. However, the efficiency of infectious virus replication was still dependent on the presence of the distal region of the M2 cytoplasmic tail. The data suggest that influenza A virus budding and genome incorporation can occur independently and provide further support for complementary roles of the M1 and M2 proteins in virus assembly.IMPORTANCE Influenza virus particle assembly involves the careful coordination of various viral and host factors to optimally produce infectious virus particles. We have previously identified a mutation at position 76 of the influenza A virus M2 protein that drastically reduces infectious virus production and filament formation with minimal effects on virus budding. In this work, we identified suppressor mutations in the M1 protein which complement this lethal M2 mutation by increasing the efficiency with which virus particles bud from infected cells and restoring filament formation at the infected-cell surface. M2 distal cytoplasmic domain sequences were still required for optimal infectivity. This indicates that M1 and M2 can functionally replace each other in some, but not all, aspects of virus particle assembly.

Discovery of berberine based derivatives as anti-influenza agent through blocking of neuraminidase

In this study, we investigated the antiviral activity of newly synthesized berberine derivatives (BD) against influenza virus infection using several strains in in vitro and in silico. The CPE reduction, pre-incubation, NA activity inhibition and molecular docking assays were used for antiviral evaluation. The anti-influenza activities of BDs were stronger than plant-derived pure commercial berberine, and some of the BDs were more potent than control drug Oseltamivir. The cytotoxicity level was observed in the range 63.16–1639μg/mL for synthesized BDs. Additionally, BDs were detected as able to block influenza viral particles. We targeted neuraminidase one of the influenza surface protein for further probing. Moreover, BDs registered competitive NA inhibition activity comparing with Oseltamivir. The active site of viral NA subunit was fully blocked by BD as the same location as Oseltamivir. The binding energies between influenza NA subunit and BD-5 were higher than Oseltamivir. More H-bonds and NA residues were occupied by BD for stronger binding ability than Oseltamivir. These results indicated that BD inhibits various strains of influenza virus by blocking of viral NA subunit.

Truncated forms of the PA protein containing only the C-terminal domains are associated with the ribonucleoprotein complex within H1N1 influenza virus particles.

We have examined the expression profile of the influenza virus PA protein in pH1N1/2009 virus-infected cells. Immunoblotting analysis of virus-infected MDCK cells revealed the presence of full-length PA protein from 8 h post-infection, together with the simultaneous appearance of PA protein species of approximately 50, 35/39 and 20/25 kDa (collectively referred to as PA*). PA* was also detected in H1N1/WSN-virus-infected cells, indicating that its presence was not virus-specific, and it was also observed in virus-infected A549 and chick embryo fibroblast (CEF) cells, indicating that its presence was not cell-type-specific. PA* was detected in cells expressing the recombinant PA protein, indicating that the PA* formation occurred in the absence of virus infection. These data collectively indicated that PA* formation is an intrinsic property of PA gene expression. The association of PA* with purified influenza virus particles was demonstrated by immunoblotting, and a protease protection assay provided evidence that PA* was packaged into virus particles. The ribonucleoprotein (RNP) complex was isolated from purified influenza virus particles using glycerol gradient centrifugation, which demonstrated that PA* was associated with the RNP complex. To the best of our knowledge, this is the first report to demonstrate that PA protein species containing only segments of the C-terminal domain form during influenza virus infection. Furthermore, these truncated PA protein species are subsequently packaged into virus particles as part of the functional RNP complex.

Mixtures of 3D disperse systems with nano- and micro-particles: Optical characterization

Multiparameter analysis of simultaneous optical data for systems of nano- and/or micro-particles (3D disperse systems, dispersions, colloids, ensembles) by presentation of system characteristics as N-dimensional vectors of optical parameters (ND-vectors) can help to elucidate changes in the state of the particles in systems. In this paper, the application of the ND-vector approach is shown on the examples of dispersion mixtures: a mixture of influenza virus particles with albumin proteins (as a model of dispersions at the process of vaccine production); a mixture of coli bacillus and clay dispersions (as natural water model). This approach can serve as an on-line control platform for the management of technological processes with mixtures.

Airborne virus detection by a sensing system using a disposable integrated impaction device

There are many respiratory infections such as influenza that cause epidemics. These respiratory infection epidemics can be effectively prevented by determining the presence or absence of infections in patients using frequent tests. We think that self-diagnosis may be possible using a system that can collect and detect biological aerosol particles in the patient’s breath because breath sampling is easy work requiring no examiner. In this paper, we report a sensing system for biological aerosol particles (SSBAP) with a disposable device. Using the system and the device, someone with no medical knowledge or skills can safely, easily, and rapidly detect infectious biological aerosol particles. The disposable device, which is the core of the SSBAP, can be an impactor for biological aerosol particles, a flow-cell for reagents, and an optical window for the fluorescent detection of collected particles. Furthermore, to detect the fluorescence of very small collected particles, this disposable device is covered with a light-blocking film that lets only fluorescence of particles pass through a fluorescence detector of the SSBAP. The SSBAP using the device can automatically detect biological aerosol particles by the following process: collecting biological aerosol particles from a patient’s breath in a sampling bag by the impaction method, labeling the collected biological aerosol particles with fluorescent dyes by the antigen–antibody reaction, removing free fluorescent dyes, and detecting the fluorescence of the biological aerosol particles. The collection efficiency of the device for microspheres aerosolized in the sampling bag was more than 97%, and the SSBAP with the device could detect more than 8.3 × 103 particles l−1 of aerosolized influenza virus particles within 10 min.

Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells.

Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7542-4) contains supplementary material, which is available to authorized users.

Mono- and polyprotic buffer systems in anion exchange chromatography of influenza virus particles

Different ions typically used in downstream processing of biologicals are evaluated for their potential in anion exchange chromatography of an industrially produced, pandemic influenza H1N1 virus. Capacity, selectivity and recovery are investigated based on single step elution parallel chromatography experiments. The inactivated H1N1 feedstream is produced in Madin-Darby Bovine Kidney cells. Interesting effects are found for sodium phosphate and sodium citrate. Both anions are triprotic kosmotropes. Anion exchange chromatography generally offers high scalability to satisfy sudden demands for vaccines, which may occur in case of an emerging influenza outbreak. Appropriate pH conditions for H1N1 adsorption are determined by Zeta potential measurements. The dynamic binding capacity of a salt tolerant polyamine-type resin is up to 6.4 times greater than the capacity of a grafted Q-type resin. Pseudo-affinity interactions of polyamines with the M2 protein of influenza may contribute to the obtained capacity increase. Both resins achieve greater capacity in sodium phosphate buffer compared to Tris/HCl. A recovery of 67% and DNA clearance close to 100% without DNAse treatment are achieved for the Q-type resin. Recovery of the virus from the salt tolerant resin requires the use of polyprotic acids in the elution buffer. 85% of the DNA and 60% of the proteins can be removed by the salt tolerant resin. The presence of sodium phosphate during anion exchange chromatography seems to support stability of the H1N1 particles in presence of hydrophobic cations.

Montmorillonite-mediated aggregation induces deformation of influenza virus particles

The interaction between influenza virus [subtype A/Puerto Rico/8/1934 H1N1, (PR8)] and montmorillonite (Mt) is investigated by transmission electron microscopy and biochemical methods to determine how PR8 morphology and viability is affected. The majority of the PR8 particles formed aggregates with the Mt. TEM analysis showed that the virus particles retained structural integrity after aggregation but exhibited changes in morphology when compared to isolated PR8 and Mt aggregated with bromelain-treated PR8 (surface glycoproteins removed). Virus deformation shows that the virions exhibit an attraction to the Mt faces, possibly through hydrophobic interaction. The mean projection area of the aggregated PR8 was (10.4±6.1)×103nm2 compared to (9.5±3.3)×103nm2 for PR8 missing the surface glycoproteins; and (8.0±3.9)×103nm2 for non-aggregated PR8 controls. The increase in projection area of the aggregated PR8 suggests that the viruses deformed to increase contact region with the Mt faces with a subsequent compression normal to the face. PR8 missing the surface protein also exhibited an increase in projection area, although to a lesser extent, indicating that both the surface glycoproteins and viral envelope are attracted to the Mt faces. Circularity calculations indicate that the aggregated PR8 (circularity: 0.69±0.16) are less round, i.e. more distorted, than either control PR8 (0.78±0.14) or aggregated PR8 without surface glycoproteins (0.76±0.12). The pleomorphic nature of influenza virus may allow it to survive the deformation induced by the Mt platelets. High resolution TEM micrographs revealed that the otherwise-round viruses flattened when in contact with platelet faces, thus increasing contact area with the Mt.The PR8 was found to remain infectious after aggregation although at a lower rate than PR8 controls. The apparent reduced infectivity is likely a result of each aggregate (containing ~102 viral particles) acting as a single infectious unit.

HCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus.

Inhibition of Influenza Virus Replication by DNA Aptamers Targeting a Cellular Component of Translation Initiation.

The genetic diversity of the influenza virus hinders the use of broad spectrum antiviral drugs and favors the appearance of resistant strains. Single-stranded DNA aptamers represent an innovative approach with potential application as antiviral compounds. The mRNAs of influenza virus possess a 5′cap structure and a 3′poly(A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins. We have studied the potential of two specific aptamers that recognize PABP1 (ApPABP7 and ApPABP11) to act as anti-influenza drugs. Both aptamers reduce viral genome expression and the production of infective influenza virus particles. The interaction of viral polymerase with the eIF4GI translation initiation factor is hindered by transfection of infected cells with both PABP1 aptamers, and ApPABP11 also inhibits the association of NS1 with PABP1 and eIF4GI. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes.

Ultrasensitive detection of influenza viruses with a glycan-based impedimetric biosensor

An ultrasensitive impedimetric glycan-based biosensor for reliable and selective detection of inactivated, but intact influenza viruses H3N2 was developed. Such glycan-based approach has a distinct advantage over antibody-based detection of influenza viruses since glycans are natural viral receptors with a possibility to selectively distinguish between potentially pathogenic influenza subtypes by the glycan-based biosensors. Build-up of the biosensor was carefully optimized with atomic force microscopy applied for visualization of the biosensor surface after binding of viruses with the topology of an individual viral particle H3N2 analyzed. The glycan biosensor could detect a glycan binding lectin with a limit of detection (LOD) of 5aM. The biosensor was finally applied for analysis of influenza viruses H3N2 with LOD of 13 viral particles in 1μl, what is the lowest LOD for analysis of influenza viral particles by the glycan-based device achieved so far. The biosensor could detect H3N2 viruses selectively with a sensitivity ratio of 30 over influenza viruses H7N7. The impedimetric biosensor presented here is the most sensitive glycan-based device for detection of influenza viruses and among the most sensitive antibody or aptamer based biosensor devices.

Influenza Virus Induces Cholesterol-Enriched Endocytic Recycling Compartments for Budozone Formation via Cell Cycle-Independent Centrosome Maturation.

Influenza virus particles are assembled at the plasma membrane in concert with incorporation of the virus genome, but the details of its spatio-temporal regulation are not understood. Here we showed that influenza virus infection induces the assembly of pericentrosomal endocytic recycling compartment (ERC) through the activation of Rab11a GTPase and cell cycle-independent maturation of centrosome by YB-1, a multifunctional protein that is involved in mitotic division, RNA metabolism and tumorigenesis. YB-1 is recruited to the centrosome in infected cells and is required for anchoring microtubules to the centrosome. We also found that viral infection accumulates cholesterol in ERC and is dependent on YB-1. Depletion of YB-1 shows reduced cholesterol-enriched ERC and prevented budozone formation at the plasma membrane. These results suggest that cholesterol in recycling endosomes, which are emanated from ERC, may trigger the virus assembly concomitantly with the packaging of the virus genome. We propose that the virus genome is transported to the plasma membrane by cholesterol-enriched recycling endosomes through cell cycle-independent activation of the centrosome by YB-1.

Use of chimeric influenza viruses as a novel internal control for diagnostic rRT-PCR assays.

Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5′ untranslated region (5′ UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BackgroundEach year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus’ fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen’s eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers.MethodsIn this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles.ResultsFor the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles.ConclusionsThis study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0152-x) contains supplementary material, which is available to authorized users.

The Usefulness of an Influenza Virus-Like Particle (VLP) Vaccine Produced in Silkworm Pupae and Virosomes and Liposomes Prepared by Chemical Means: From Virosome to VLP and the Future of Vaccines

In our quest for an ideal influenza vaccine in 1989, we successfully prepared a muramyl dipeptide (MDP)-liposome-containing influenza virus hemagglutinin (HA) and neuraminidase (NA) virosome vaccine. This MDP-virosome vaccine showed enhanced HA immunogenicity in mice and elicited superior antibody production in humans, and a high HI titer was retained for up to 2 years. Generation of a cellular immune response was also confirmed by the production of cytotoxic T lymphocytes in mice. In fact, these killer T cells reduced the titer of the H3N2, H1N1 and B virus in the lungs of infected mice. Based on the finding that this MDP-virosome vaccine could elicit both humoral and cellular immune responses (i.e., high antibody activity and killer T cells), which is a requirement for an ideal vaccine, we examined the possibility of preparing the VLP vaccine in silkworms. Here, we show that significant amounts of influenza VLPs could be successfully produced in silkworm pupae by using a chimeric (multiple hybrid) HA DNA. In fact, the HA titers in homogenates from the infected pupae reached a mean value of 0.8 million HA units (HAU), equivalent to approximately 2,000 μg of HA protein per pupa, which is more than 50-fold higher than that produced in embryonated chicken eggs. Analysis of the VLPs in the purified HA fractions showed that they were attractive structures ranging from 30 to 300 nm in diameter. The spikes on the surface of the VLPs were approximately 14 nm long, densely packed, and were very similar to those on actual influenza virus particles. Subsequent important evidence was symbolized in the following genome constellation. In the present study, we showed, for the first time, that VLPs could be synthesized in silkworms by injection of a single synthetic chimeric HA DNA. These VLPs reflected the characteristic structure of the influenza virosome and elicited immune responses that were much higher than those elicited by the MDP-liposome prepared in our previous study. Therefore, the use of chemical immunomodulators such as MDP should be considered in the future for the development of VLP vaccines.

Actin-myosin network is required for proper assembly of influenza virus particles

Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation.

Particle quantification of influenza viruses by high performance liquid chromatography

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5M NaCl salt with 20mM Tris–HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5min. A linear response range was established between 1×109 and 1×1011 virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07×108 and 4.35×109 whereas the limit of quantification was between 6.90×108 and 1.45×1010VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.