Single-particle measurements of filamentous influenza virions reveal damage induced by freezing

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to −70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections.

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Single-particle measurements of filamentous influenza virions reveal damage induced by freezing

10.1101/686642 ◽

2019 ◽

Author(s):

Jack C. Hirst ◽

Edward C. Hutchinson

Keyword(s):

Influenza Virus ◽

Immunofluorescence Microscopy ◽

Influenza Viruses ◽

Virus Infections ◽

Median Length ◽

Minimal Impact ◽

Particle Measurements ◽

Functional Studies ◽

Laboratory Techniques ◽

Virus Produce

AbstractClinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantity and characterise the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70°C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced both their concentration and median length. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by using the cryoprotectant DMSO. We recommend that functional studies of filaments characterise virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterisations of filaments that are required to understand their roles in natural influenza virus infections.

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Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

Scientific Reports ◽

10.1038/s41598-020-79590-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James D. Allen ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Next Generation ◽

Virus Infections ◽

Wild Type ◽

Influenza Hemagglutinin ◽

H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

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Sequential Seasonal H1N1 Influenza Virus Infections Protect Ferrets against Novel 2009 H1N1 Influenza Virus

Journal of Virology ◽

10.1128/jvi.02257-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1400-1410 ◽

Cited By ~ 45

Author(s):

Donald M. Carter ◽

Chalise E. Bloom ◽

Eduardo J. M. Nascimento ◽

Ernesto T. A. Marques ◽

Jodi K. Craigo ◽

...

Keyword(s):

Influenza Virus ◽

H1n1 Influenza ◽

Cross Reactivity ◽

Influenza Viruses ◽

The Novel ◽

Virus Infections ◽

H1n1 Influenza Virus ◽

Virus Shedding ◽

2009 H1n1 ◽

Novel H1n1 Influenza

ABSTRACTIndividuals <60 years of age had the lowest incidence of infection, with ∼25% of these people having preexisting, cross-reactive antibodies to novel 2009 H1N1 influenza. Many people >60 years old also had preexisting antibodies to novel H1N1. These observations are puzzling because the seasonal H1N1 viruses circulating during the last 60 years were not antigenically similar to novel H1N1. We therefore hypothesized that a sequence of exposures to antigenically different seasonal H1N1 viruses can elicit an antibody response that protects against novel 2009 H1N1. Ferrets were preinfected with seasonal H1N1 viruses and assessed for cross-reactive antibodies to novel H1N1. Serum from infected ferrets was assayed for cross-reactivity to both seasonal and novel 2009 H1N1 strains. These results were compared to those of ferrets that were sequentially infected with H1N1 viruses isolated prior to 1957 or more-recently isolated viruses. Following seroconversion, ferrets were challenged with novel H1N1 influenza virus and assessed for viral titers in the nasal wash, morbidity, and mortality. There was no hemagglutination inhibition (HAI) cross-reactivity in ferrets infected with any single seasonal H1N1 influenza viruses, with limited protection to challenge. However, sequential H1N1 influenza infections reduced the incidence of disease and elicited cross-reactive antibodies to novel H1N1 isolates. The amount and duration of virus shedding and the frequency of transmission following novel H1N1 challenge were reduced. Exposure to multiple seasonal H1N1 influenza viruses, and not to any single H1N1 influenza virus, elicits a breadth of antibodies that neutralize novel H1N1 even though the host was never exposed to the novel H1N1 influenza viruses.

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Historical H1N1 Influenza Virus Imprinting Increases Vaccine Protection by Influencing the Activity and Sustained Production of Antibodies Elicited at Vaccination in Ferrets

Vaccines ◽

10.3390/vaccines7040133 ◽

2019 ◽

Vol 7 (4) ◽

pp. 133 ◽

Cited By ~ 7

Author(s):

Magen E. Francis ◽

Mara McNeil ◽

Nicholas J. Dawe ◽

Mary K. Foley ◽

Morgan L. King ◽

...

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Time Course ◽

Recovery Period ◽

Haemagglutination Inhibition ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Immune Memory ◽

Sublethal Dose ◽

Virus Infections

Influenza virus imprinting is now understood to significantly influence the immune responses and clinical outcome of influenza virus infections that occur later in life. Due to the yearly cycling of influenza viruses, humans are imprinted with the circulating virus of their birth year and subsequently build a complex influenza virus immune history. Despite this knowledge, little is known about how the imprinting strain influences vaccine responses. To investigate the immune responses of the imprinted host to split-virion vaccination, we imprinted ferrets with a sublethal dose of the historical seasonal H1N1 strain A/USSR/90/1977. After a +60-day recovery period to build immune memory, ferrets were immunized and then challenged on Day 123. Antibody specificity and recall were investigated throughout the time course. At challenge, the imprinted vaccinated ferrets did not experience significant disease, while naïve-vaccinated ferrets had significant weight loss. Haemagglutination inhibition assays showed that imprinted ferrets had a more robust antibody response post vaccination and increased virus neutralization activity. Imprinted-vaccinated animals had increased virus-specific IgG antibodies compared to the other experimental groups, suggesting B-cell maturity and plasticity at vaccination. These results should be considered when designing the next generation of influenza vaccines.

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Association of Severe Influenza Virus Infections With CD226 (DNAM-1) Variants

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz270 ◽

2019 ◽

Vol 220 (7) ◽

pp. 1162-1165 ◽

Cited By ~ 2

Author(s):

Monika Redlberger-Fritz ◽

Hannes Vietzen ◽

Elisabeth Puchhammer-Stöckl

Keyword(s):

Influenza Virus ◽

Intensive Care ◽

Cell Response ◽

Nk Cell ◽

Virus Disease ◽

Cell Receptor ◽

Influenza Viruses ◽

Virus Infections ◽

Severe Influenza ◽

Nk Cell Receptor

Abstract Natural killer (NK)-cell response against influenza viruses partly depends on expression of CD112, a ligand for NK-cell receptor CD226 (DNAM-1). We analyzed whether particular CD226 variants were associated with influenza disease severity. Comparison between 145 patients hospitalized with severe influenza at intensive care units (ICU) with 139 matched influenza-positive outpatients showed that presence of the rs763362 G allele (GG, AG) was associated with occurrence of severe influenza infections (P = .0076). Also, a higher frequency of rs727088 G and rs763361 T alleles was observed in the ICU group. Thus, CD226 variants may contribute to the severity of influenza virus disease.

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Influenza A Virus Vaccination: Immunity, Protection, and Recent Advances Toward A Universal Vaccine

Vaccines ◽

10.3390/vaccines8030434 ◽

2020 ◽

Vol 8 (3) ◽

pp. 434 ◽

Cited By ~ 2

Author(s):

Christopher E. Lopez ◽

Kevin L. Legge

Keyword(s):

Immune Response ◽

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Virus Infections ◽

Universal Vaccine

Influenza virus infections represent a serious public health threat and account for significant morbidity and mortality worldwide due to seasonal epidemics and periodic pandemics. Despite being an important countermeasure to combat influenza virus and being highly efficacious when matched to circulating influenza viruses, current preventative strategies of vaccination against influenza virus often provide incomplete protection due the continuous antigenic drift/shift of circulating strains of influenza virus. Prevention and control of influenza virus infection with vaccines is dependent on the host immune response induced by vaccination and the various vaccine platforms induce different components of the local and systemic immune response. This review focuses on the immune basis of current (inactivated influenza vaccines (IIV) and live attenuated influenza vaccines (LAIV)) as well as novel vaccine platforms against influenza virus. Particular emphasis will be placed on how each platform induces cross-protection against heterologous influenza viruses, as well as how this immunity compares to and contrasts from the “gold standard” of immunity generated by natural influenza virus infection.

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Immunization with Live Attenuated Influenza Viruses That Express Altered NS1 Proteins Results in Potent and Protective Memory CD8+ T-Cell Responses

Journal of Virology ◽

10.1128/jvi.01317-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1847-1855 ◽

Cited By ~ 31

Author(s):

Scott N. Mueller ◽

William A. Langley ◽

Elena Carnero ◽

Adolfo García-Sastre ◽

Rafi Ahmed

Keyword(s):

Influenza Virus ◽

T Cell ◽

B Cell ◽

Protective Immunity ◽

T Cell Responses ◽

Influenza Viruses ◽

Wild Type Virus ◽

Virus Infections ◽

Cell Responses ◽

B Cell Responses

ABSTRACT The generation of vaccines that induce long-lived protective immunity against influenza virus infections remains a challenging goal. Ideally, vaccines should elicit effective humoral and cellular immunity to protect an individual from infection or disease. Cross-reactive T- and B-cell responses that are elicited by live virus infections may provide such broad protection. Optimal induction of T-cell responses involves the action of type I interferons (IFN-I). Influenza virus expressed nonstructural protein 1 (NS1) functions as an inhibitor of IFN-I and promotes viral growth. We wanted to examine the priming of CD8+ T-cell responses to influenza virus in the absence of this inhibition of IFN-I production. We generated recombinant mouse-adapted influenza A/PR/8/34 viruses with NS1 truncations and/or deletions that also express the gp33-41 epitope from lymphocytic choriomeningitis virus. Intranasal infection of mice with the attenuated viruses primed long-lived T- and B-cell responses despite significantly reduced viral replication in the lungs compared to wild-type virus. Antigen-specific CD8+ T cells expanded upon rechallenge and generated increased protective memory T-cell populations after boosting. These results show that live attenuated influenza viruses expressing truncated NS1 proteins can prime protective immunity and may have implications for the design of novel modified live influenza virus vaccines.

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Activity of enisamium, an isonicotinic acid derivative, against influenza viruses in differentiated normal human bronchial epithelial cells

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206618811416 ◽

2018 ◽

Vol 26 ◽

pp. 204020661881141 ◽

Cited By ~ 1

Author(s):

David Boltz ◽

Xinjian Peng ◽

Miguel Muzzio ◽

Pradyot Dash ◽

Paul G Thomas ◽

...

Keyword(s):

Influenza Virus ◽

Epithelial Cells ◽

Antiviral Activity ◽

Bronchial Epithelial Cells ◽

Influenza Viruses ◽

Virus Infections ◽

Normal Human Bronchial Epithelial ◽

Human Bronchial Epithelial Cells ◽

Bronchial Epithelial ◽

Normal Human

Aims New therapeutics for the control of influenza virus infections are needed to alleviate the burden caused by seasonal epidemics and occasional pandemics, and to overcome the potential risk of drug-resistance emergence. Enisamium iodide (Amizon®, Farmak) is currently approved for clinical use for the treatment of influenza in 11 countries which includes Ukraine, Russia, Belarus, Kazakhstan, and Uzbekistan. However, experimental evidence of the antiviral activity of enisamium has not been reported. Methods Antiviral activity of enisamium was assessed by virus yield reduction assays using differentiated normal human bronchial epithelial cells. Permeability of enisamium into differentiated normal human bronchial epithelial cells and its cytotoxicity were also assessed, and comparisons with other cell lines were made. Results Enisamium inhibited replication of multiple subtypes of influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, the zoonotic H5N1 and H7N9, neuraminidase inhibitor-resistant variant carrying the H275Y NA substitution (N1 numbering), and influenza B virus at doses 23- to 64-fold lower than cytotoxic concentrations. The permeability of enisamium in Madin–Darby canine kidney cells (where no antiviral activity was found) was less than 0.08%, while higher permeability was observed in differentiated normal human bronchial epithelial cells (1.9%). The kinetics of enisamium intracellular uptake in differentiated normal human bronchial epithelial cells was concentration dependent. In time-of-addition experiments in differentiated normal human bronchial epithelial cells, enisamium treatment within 4 h after A(H1N1) virus inoculation resulted in 100-fold or greater reductions in virus titers, suggesting that it affects an early stage of the virus life cycle. Conclusions Enisamium exhibits antiviral activity against influenza viruses in vitro, supporting the reported clinical efficacy against influenza virus infections.

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Ethanol Extract of Caesalpinia decapetala Inhibits Influenza Virus Infection In Vitro and In Vivo

Viruses ◽

10.3390/v12050557 ◽

2020 ◽

Vol 12 (5) ◽

pp. 557 ◽

Cited By ~ 1

Author(s):

Li Zhang ◽

Jungang Chen ◽

Chang Ke ◽

Haiwei Zhang ◽

Shoujun Zhang ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Antiviral Agents ◽

Influenza Virus Infection ◽

Virus Titer ◽

Ethanol Extract ◽

Influenza Viruses ◽

Virus Infections

Influenza virus infections can lead to viral pneumonia and acute respiratory distress syndrome in severe cases, causing significant morbidity and mortality and posing a great threat to human health. Because of the diversity of influenza virus strains and drug resistance to the current direct antiviral agents, there have been no effective drugs as yet to cure all patients infected by influenza viruses. Natural products from plants contain compounds with diverse structures that have the potential to interact with multiple host and virus factors. In this study, we identified the ethanol extract of Caesalpinia decapetala (Roth) Alston (EEC) as an inhibitor against the replication of a panel of influenza A and B viruses both on human pulmonary epithelial A549 and human monocytic U937 cells. The animal study revealed that EEC administration reduces the weight loss and improves the survival rate of mice infected with lethal influenza virus. Also, EEC treatment attenuated lung injury and reduced virus titer significantly. In conclusion, we showed that EEC has antiviral activity both in vitro and in vivo, suggesting that the plant C. decapetala has the potential to be further developed as a resource of new anti-influenza drugs.

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Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00781-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6007-6016 ◽

Cited By ~ 8

Author(s):

Alice W. Tsai ◽

Colleen F. McNeil ◽

Joshua R. Leeman ◽

Hamilton B. Bennett ◽

Kwame Nti-Addae ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Therapeutic Agents ◽

Influenza Viruses ◽

Antigenic Drift ◽

Whole Body ◽

Infection Model ◽

Virus Infections

ABSTRACTThrough antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellentin vitroandin vivoproperties have been identified. To evaluate thein vivoefficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

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