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Perry SS, Brice DC, Sakr AA, Kandeil A, DeBeauchamp J, Ghonim M, Jones J, Miller L, Vegesana K, Crawford JC, Langfitt DM, Kercher L, Abdelsamed HA, Webster RG, Thomas PG, Webby RJ, Okda FA. Modulation of cytokeratin and cytokine/chemokine expression following influenza virus infection of differentiated human tonsillar epithelial cells. J Virol 2025; 99:e0146024. [PMID: 39791909 PMCID: PMC11852761 DOI: 10.1128/jvi.01460-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Accepted: 12/08/2024] [Indexed: 01/12/2025] Open
Abstract
The tonsils have been identified as a site of replication for Epstein-Barr virus, adenovirus, human papillomavirus, and other respiratory viruses. Human tonsil epithelial cells (HTECs) are a heterogeneous group of actively differentiating cells. Here, we investigated the cellular features and susceptibility of differentiated HTECs to specific influenza viruses, including expression of avian-type and mammalian-type sialic acid (SA) receptors, viral replication dynamics, and the associated cytokine secretion profiles. We found that differentiated HTECs possess more abundant α2,3-linked SA (preferentially bound by avian influenza viruses) than α2,6-linked SA (preferentially bound by mammalian strains). This dual receptor expression suggests a role in influenza virus adaptation and tropism within the tonsils by facilitating the binding and entry of multiple influenza virus strains. Our results indicated the susceptibility of differentiated HTECs to a wide range of influenza viruses from human, swine, and avian hosts. Virus production for most strains was detected as early as 1 day post-infection (dpi), and typically peaked by 3 dpi. However, pandemic H1N1 virus showed remarkably delayed replication kinetics that did not peak until at least 7 dpi. Notably, influenza virus infection impacted the expression of cytokeratins in HTEC cultures, which correlated with altered cytokine secretion patterns. These patterns varied within the strains but were most distinct in swine H3N2 infection. In conclusion, differentiated HTECs exhibited a strain-specific pattern of influenza virus replication and innate immune responses that included changes in cytokeratin and cytokine expression. These studies shed light on the complex interplay between influenza viruses and host cells in the tonsils. IMPORTANCE To develop effective interventions against influenza, it is important to identify host factors affecting pathogenesis and immune responses. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and the immune response. Influenza virus tropism in the human upper respiratory tract is a key determinant of host-range, pathogenesis, and transmission. Accordingly, experimental models using primary cells from the human respiratory tract are relevant for assessing virus tropism and replication competence. Our study addresses the dynamics of influenza virus replication in HTECs, including cellular tropism, infectivity, and cytokeratin and cytokine expression. The results of this study highlight the complex interplay between structural proteins and immune signaling pathways, all of which provide valuable insights into host-virus interactions.
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Affiliation(s)
- S. Scott Perry
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - David C. Brice
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Ahmed Atef Sakr
- Cornell Veterinary Biobank, Cornell University College of Veterinary Medicine, Ithaca, New York, USA
| | - Ahmed Kandeil
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
- National Research Center, Giza, Egypt
| | - Jennifer DeBeauchamp
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Mohamed Ghonim
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Jeremy Jones
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Lance Miller
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Kasi Vegesana
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Jeremy Chase Crawford
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Deanna M. Langfitt
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Lisa Kercher
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Hossam A. Abdelsamed
- Immunology Center of Georgia (IMMCG), Department of Physiology, Medical College of Georgia (MCG), Augusta University, Augusta, Georgia, USA
| | - Robert G. Webster
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Paul G. Thomas
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Richard J. Webby
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Faten A. Okda
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
- National Research Center, Giza, Egypt
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Luo R, Lv C, Wang T, Deng X, Sima M, Guo J, Qi J, Sun W, Shen B, Li Y, Yue D, Gao Y. A potential Chinese medicine monomer against influenza A virus and influenza B virus: isoquercitrin. Chin Med 2023; 18:144. [PMID: 37919750 PMCID: PMC10621105 DOI: 10.1186/s13020-023-00843-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 09/27/2023] [Indexed: 11/04/2023] Open
Abstract
BACKGROUND Influenza viruses, especially Influenza A virus and Influenza B virus, are respiratory pathogens and can cause seasonal epidemics and pandemics. Severe influenza viruses infection induces strong host-defense response and excessive inflammatory response, resulting in acute lung damage, multiple organ failure and high mortality. Isoquercitrin is a Chinese medicine monomer, which was reported to have multiple biological activities, including antiviral activity against HSV, IAV, SARS-CoV-2 and so on. Aims of this study were to assess the in vitro anti-IAV and anti-IBV activity, evaluate the in vivo protective efficacy against lethal infection of the influenza virus and searched for the more optimal method of drug administration of isoquercitrin. METHODS In vitro infection model (MDCK and A549 cells) and mouse lethal infection model of Influenza A virus and Influenza B virus were used to evaluate the antiviral activity of isoquercitrin. RESULTS Isoquercitrin could significantly suppress the replication in vitro and in vivo and reduced the mortality of mouse lethal infection models. Compared with virus infection group, isoquercitrin mitigated lung and multiple organ damage. Moreover, isoquercitrin blocked hyperproduction of cytokines induced by virus infection via inactivating NF-κB signaling. Among these routes of isoquercitrin administration, intramuscular injection is a better drug delivery method. CONCLUSION Isoquercitrin is a potential Chinese medicine monomer Against Influenza A Virus and Influenza B Virus infection.
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Affiliation(s)
- Rongbo Luo
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
| | - Chaoxiang Lv
- The Research Center for Preclinical Medicine, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Tiecheng Wang
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Xiuwen Deng
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
- College of Integrated Chinese and Western Medicine, Changchun University of Chinese Medicine, Changchun, Jilin, 130117, China
| | - Mingwei Sima
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
- College of Integrated Chinese and Western Medicine, Changchun University of Chinese Medicine, Changchun, Jilin, 130117, China
| | - Jin Guo
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
- College of Life Sciences, Shandong Normal University, Jinan, 250014, China
| | - Jing Qi
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
- College of Life Sciences, Northeast Normal University, Changchun, 130021, China
| | - Weiyang Sun
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Beilei Shen
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
| | - Yuanguo Li
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China
| | - Donghui Yue
- School of Medical Sciences, Changchun University of Chinese Medicine, Changchun, Jilin, 130117, China.
| | - Yuwei Gao
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China.
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China.
- College of Integrated Chinese and Western Medicine, Changchun University of Chinese Medicine, Changchun, Jilin, 130117, China.
- College of Life Sciences, Shandong Normal University, Jinan, 250014, China.
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Golikov MV, Bartosch B, Smirnova OA, Ivanova ON, Ivanov AV. Plasma-Like Culture Medium for the Study of Viruses. mBio 2023; 14:e0203522. [PMID: 36515528 PMCID: PMC9973327 DOI: 10.1128/mbio.02035-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Viral infections attract more and more attention, especially after the emergence of novel zoonotic coronaviruses and the monkeypox virus over the last 2 decades. Research on viruses is based to a great extent on mammalian cell lines that are permissive to the respective viruses. These cell lines are usually cultivated according to the protocols established in the 1950s to 1970s, although it is clear that classical media have a significant imprint on cell growth, phenotype, and especially metabolism. So, recently in the field of biochemistry and metabolomics novel culture media have been developed that resemble human blood plasma. As perturbations in metabolic and redox pathways during infection are considered significant factors of viral pathogenesis, these novel medium formulations should be adapted by the virology field. So far, there are only scarce data available on viral propagation efficiencies in cells cultivated in plasma-like media. But several groups have presented convincing data on the use of such media for cultivation of uninfected cells. The aim of the present review is to summarize the current state of research in the field of plasma-resembling culture media and to point out the influence of media on various cellular processes in uninfected cells that may play important roles in viral replication and pathogenesis in order to sensitize virology research to the use of such media.
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Affiliation(s)
- Mikhail V. Golikov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Birke Bartosch
- Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, Lyon, France
| | - Olga A. Smirnova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Olga N. Ivanova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Alexander V. Ivanov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
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Vandoorn E, Stadejek W, Leroux-Roels I, Leroux-Roels G, Parys A, Van Reeth K. Human Immunity and Susceptibility to Influenza A(H3) Viruses of Avian, Equine, and Swine Origin. Emerg Infect Dis 2023; 29:98-109. [PMID: 36573615 PMCID: PMC9796212 DOI: 10.3201/eid2901.220943] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Influenza A viruses (IAVs) of subtype H3 that infect humans are antigenically divergent from those of birds, horses, and swine. Human immunity against these viruses might be limited, implying potential pandemic risk. To determine human risk, we selected 4 avian, 1 equine, and 3 swine IAVs representing major H3 lineages. We tested serum collected during 2017-2018 from 286 persons in Belgium for hemagglutination inhibiting antibodies and virus neutralizing antibodies against those animal-origin IAVs and tested replication in human airway epithelia. Seroprevalence rates for circulating IAVs from swine in North America were >51%, swine in Europe 7%-37%, and birds and equids ≤12%. Replication was efficient for cluster IV-A IAVs from swine in North America and IAVs from swine in Europe, intermediate for IAVs from horses and poultry, and absent for IAVs from wild birds and a novel human-like swine IAV in North America. Public health risk may be highest for swine H3 IAVs.
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Influenza A virus strain PR/8/34, but neither HAM/2009 nor WSN/33, is transiently inhibited by the PB2-targeting drug paliperidone. Arch Virol 2023; 168:63. [PMID: 36637551 PMCID: PMC9839214 DOI: 10.1007/s00705-022-05696-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Accepted: 12/01/2022] [Indexed: 01/14/2023]
Abstract
Influenza A virus (FLUAV) is a significant human pathogen. In silico structural analysis (PMID 28628827) has suggested that the FDA-approved drug paliperidone interferes with the binding of the FLUAV polymerase subunit PB2 to the nucleoprotein NP. We found that paliperidone inhibits FLUAV A/PR/8/34 early after infection of canine MDCK II, human A549, and human primary bronchial cells, but not at late time points. No effect was detectable against the strains A/Hamburg/05/2009 and A/WSN/33. Moreover, paliperidone indeed disturbed the interaction between the PB2 and the NP of A/PR/8/34 and reduced early viral RNA and protein synthesis by approximately 50%. Thus, paliperidone has measurable but transient and virus-strain-restricted effects on FLUAV.
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Muralidharan A, Gravel C, Harris G, Hashem AM, Zhang W, Safronetz D, Van Domselaar G, Krammer F, Sauve S, Rosu-Myles M, Wang L, Chen W, Li X. Universal antibody targeting the highly conserved fusion peptide provides cross-protection in mice. Hum Vaccin Immunother 2022; 18:2083428. [PMID: 35724343 PMCID: PMC9621047 DOI: 10.1080/21645515.2022.2083428] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Influenza is a major public health concern causing millions of hospitalizations every year. The current vaccines need annual updating based on prediction of likely strains in the upcoming season. However, mismatches between vaccines and the actual circulating viruses can occur, reducing vaccine effectiveness significantly because of the remarkably high rate of mutation in the viral glycoprotein, hemagglutinin (HA). Clearly, it would be of great interest to determine the potential role of universally conserved epitopes in inducing protective immunity. Here, an antibody against the 14-aa fusion peptide sequence at the N-terminus of the HA2 subunit (Uni-1) was investigated for its ability to elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro and cross-protection against lethal infection in animals. Uni-1, known to neutralize influenza type A (IAV) in vitro, was found to induce strong ADCC against diverse influenza viruses, including human and avian IAVs and both lineages of type B (IBV). The ADCC effects against human IAVs by Uni-1 was comparable to ADCC induced by well-characterized antibodies, F10 and FI6V3. Importantly, mice treated with Uni-1 were protected against lethal challenge of IAV and IBV. These results revealed the versatile effector functions of this universal antibody against markedly diverse strains of both IAV and IBV.
The fusion peptide is the only universally conserved epitope in both IAV and IBV Mono-specific universal antibody induces strong ADCC against human and avian IAV Mono-specific universal antibody induces strong ADCC against IBV from both genetic lineages of IBV The antibody has bi-functional effector functions against several influenza viruses
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Affiliation(s)
- Abenaya Muralidharan
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.,Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, USA
| | - Caroline Gravel
- Centre for Biologics Evaluation, Biologic and Radiopharmaceutical Drugs Directorate, HPFB, Health Canada and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Ottawa, Canada
| | - Greg Harris
- Human Health Therapeutics (HHT) Research Center, National Research Council of Canada, Ottawa, Canada
| | - Anwar M Hashem
- Vaccines and Immunotherapy Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia.,Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Wanyue Zhang
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.,Centre for Biologics Evaluation, Biologic and Radiopharmaceutical Drugs Directorate, HPFB, Health Canada and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Ottawa, Canada
| | - David Safronetz
- National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada
| | - Gary Van Domselaar
- National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada
| | - Florian Krammer
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, USA
| | - Simon Sauve
- Centre for Biologics Evaluation, Biologic and Radiopharmaceutical Drugs Directorate, HPFB, Health Canada and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Ottawa, Canada
| | - Michael Rosu-Myles
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.,Centre for Biologics Evaluation, Biologic and Radiopharmaceutical Drugs Directorate, HPFB, Health Canada and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Ottawa, Canada
| | - Lisheng Wang
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada
| | - Wangxue Chen
- Human Health Therapeutics (HHT) Research Center, National Research Council of Canada, Ottawa, Canada
| | - Xuguang Li
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.,Centre for Biologics Evaluation, Biologic and Radiopharmaceutical Drugs Directorate, HPFB, Health Canada and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Ottawa, Canada
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Rattanaburi S, Sawaswong V, Nimsamer P, Mayuramart O, Sivapornnukul P, Khamwut A, Chanchaem P, Kongnomnan K, Suntronwong N, Poovorawan Y, Payungporn S. Genome characterization and mutation analysis of human influenza A virus in Thailand. Genomics Inform 2022; 20:e21. [PMID: 35794701 PMCID: PMC9299564 DOI: 10.5808/gi.21077] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 01/24/2022] [Accepted: 04/05/2022] [Indexed: 11/20/2022] Open
Abstract
The influenza A viruses have high mutation rates and cause a serious health problem worldwide. Therefore, this study focused on genome characterization of the viruses isolated from Thai patients based on the next-generation sequencing technology. The nasal swabs were collected from patients with influenza-like illness in Thailand during 2017-2018. Then, the influenza A viruses were detected by reverse transcription-quantitative polymerase chain reaction and isolated by MDCK cells. The viral genomes were amplified and sequenced by Illumina MiSeq platform. Whole genome sequences were used for characterization, phylogenetic construction, mutation analysis and nucleotide diversity of the viruses. The result revealed that 90 samples were positive for the viruses including 44 of A/ H1N1 and 46 of A/H3N2. Among these, 43 samples were successfully isolated and then the viral genomes of 25 samples were completely amplified. Finally, 17 whole genomes of the viruses (A/H1N1, n=12 and A/H3N2, n=5) were successfully sequenced with an average of 232,578 mapped reads and 1,720 genome coverage per sample. Phylogenetic analysis demonstrated that the A/H1N1 viruses were distinguishable from the recommended vaccine strains. However, the A/H3N2 viruses from this study were closely related to the recommended vaccine strains. The nonsynonymous mutations were found in all genes of both viruses, especially in hemagglutinin (HA) and neuraminidase (NA) genes. The nucleotide diversity analysis revealed negative selection in the PB1, PA, HA, and NA genes of the A/H1N1 viruses. High-throughput data in this study allow for genetic characterization of circulating influenza viruses which would be crucial for preparation against pandemic and epidemic outbreaks in the future.
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Affiliation(s)
- Somruthai Rattanaburi
- Interdisciplinary Program of Biomedical Sciences, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Vorthon Sawaswong
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Pattaraporn Nimsamer
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Oraphan Mayuramart
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Pavaret Sivapornnukul
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
- Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Ariya Khamwut
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Prangwalai Chanchaem
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Kritsada Kongnomnan
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Nungruthai Suntronwong
- Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Yong Poovorawan
- Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Sunchai Payungporn
- Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
- Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
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Broad-Spectrum Antiviral Activity of the Amphibian Antimicrobial Peptide Temporin L and Its Analogs. Int J Mol Sci 2022; 23:ijms23042060. [PMID: 35216177 PMCID: PMC8878748 DOI: 10.3390/ijms23042060] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 02/08/2022] [Accepted: 02/11/2022] [Indexed: 12/04/2022] Open
Abstract
The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro3, DLeu9 TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.
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Rijsbergen LC, van Dijk LLA, Engel MFM, de Vries RD, de Swart RL. In Vitro Modelling of Respiratory Virus Infections in Human Airway Epithelial Cells - A Systematic Review. Front Immunol 2021; 12:683002. [PMID: 34489934 PMCID: PMC8418200 DOI: 10.3389/fimmu.2021.683002] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 07/30/2021] [Indexed: 12/12/2022] Open
Abstract
Respiratory tract infections (RTI) are a major cause of morbidity and mortality in humans. A large number of RTIs is caused by viruses, often resulting in more severe disease in infants, elderly and the immunocompromised. Upon viral infection, most individuals experience common cold-like symptoms associated with an upper RTI. However, in some cases a severe and sometimes life-threatening lower RTI may develop. Reproducible and scalable in vitro culture models that accurately reflect the human respiratory tract are needed to study interactions between respiratory viruses and the host, and to test novel therapeutic interventions. Multiple in vitro respiratory cell culture systems have been described, but the majority of these are based on immortalized cell lines. Although useful for studying certain aspects of viral infections, such monomorphic, unicellular systems fall short in creating an understanding of the processes that occur at an integrated tissue level. Novel in vitro models involving primary human airway epithelial cells and, more recently, human airway organoids, are now in use. In this review, we describe the evolution of in vitro cell culture systems and their characteristics in the context of viral RTIs, starting from advances after immortalized cell cultures to more recently developed organoid systems. Furthermore, we describe how these models are used in studying virus-host interactions, e.g. tropism and receptor studies as well as interactions with the innate immune system. Finally, we provide an outlook for future developments in this field, including co-factors that mimic the microenvironment in the respiratory tract.
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Affiliation(s)
- Laurine C. Rijsbergen
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Laura L. A. van Dijk
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Maarten F. M. Engel
- Medical Library, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Rory D. de Vries
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Rik L. de Swart
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
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Gard AL, Luu RJ, Miller CR, Maloney R, Cain BP, Marr EE, Burns DM, Gaibler R, Mulhern TJ, Wong CA, Alladina J, Coppeta JR, Liu P, Wang JP, Azizgolshani H, Fezzie RF, Balestrini JL, Isenberg BC, Medoff BD, Finberg RW, Borenstein JT. High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro. Sci Rep 2021; 11:14961. [PMID: 34294757 PMCID: PMC8298517 DOI: 10.1038/s41598-021-94095-7] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Accepted: 07/01/2021] [Indexed: 12/15/2022] Open
Abstract
Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.
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Affiliation(s)
- A L Gard
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - R J Luu
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - C R Miller
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - R Maloney
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - B P Cain
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - E E Marr
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - D M Burns
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - R Gaibler
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - T J Mulhern
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - C A Wong
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - J Alladina
- Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, USA
| | - J R Coppeta
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - P Liu
- Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
| | - J P Wang
- Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
| | - H Azizgolshani
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | | | - J L Balestrini
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - B C Isenberg
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA
| | - B D Medoff
- Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, USA
| | - R W Finberg
- Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
| | - J T Borenstein
- Bioengineering Division, Draper, Cambridge, MA, 02139, USA.
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11
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Animal Models Utilized for the Development of Influenza Virus Vaccines. Vaccines (Basel) 2021; 9:vaccines9070787. [PMID: 34358203 PMCID: PMC8310120 DOI: 10.3390/vaccines9070787] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Revised: 07/08/2021] [Accepted: 07/10/2021] [Indexed: 12/25/2022] Open
Abstract
Animal models have been an important tool for the development of influenza virus vaccines since the 1940s. Over the past 80 years, influenza virus vaccines have evolved into more complex formulations, including trivalent and quadrivalent inactivated vaccines, live-attenuated vaccines, and subunit vaccines. However, annual effectiveness data shows that current vaccines have varying levels of protection that range between 40–60% and must be reformulated every few years to combat antigenic drift. To address these issues, novel influenza virus vaccines are currently in development. These vaccines rely heavily on animal models to determine efficacy and immunogenicity. In this review, we describe seasonal and novel influenza virus vaccines and highlight important animal models used to develop them.
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12
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Luczo JM, Bousse T, Johnson SK, Jones CA, Pearce N, Neiswanger CA, Wang MX, Miller EA, Petrovsky N, Wentworth DE, Bronshtein V, Papania M, Tompkins SM. Intranasal powder live attenuated influenza vaccine is thermostable, immunogenic, and protective against homologous challenge in ferrets. NPJ Vaccines 2021; 6:59. [PMID: 33883559 PMCID: PMC8060263 DOI: 10.1038/s41541-021-00320-9] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Accepted: 03/10/2021] [Indexed: 02/06/2023] Open
Abstract
Influenza viruses cause annual seasonal epidemics and sporadic pandemics; vaccination is the most effective countermeasure. Intranasal live attenuated influenza vaccines (LAIVs) are needle-free, mimic the natural route of infection, and elicit robust immunity. However, some LAIVs require reconstitution and cold-chain requirements restrict storage and distribution of all influenza vaccines. We generated a dry-powder, thermostable LAIV (T-LAIV) using Preservation by Vaporization technology and assessed the stability, immunogenicity, and efficacy of T-LAIV alone or combined with delta inulin adjuvant (Advax™) in ferrets. Stability assays demonstrated minimal loss of T-LAIV titer when stored at 25 °C for 1 year. Vaccination of ferrets with T-LAIV alone or with delta inulin adjuvant elicited mucosal antibody and robust serum HI responses in ferrets, and was protective against homologous challenge. These results suggest that the Preservation by Vaporization-generated dry-powder vaccines could be distributed without refrigeration and administered without reconstitution or injection. Given these significant advantages for vaccine distribution and delivery, further research is warranted.
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Affiliation(s)
- Jasmina M Luczo
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA
- Emory-UGA Centers of Excellence for Influenza Research and Surveillance (CEIRS), Athens, Georgia, 30602, USA
| | - Tatiana Bousse
- Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
| | - Scott K Johnson
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA
| | - Cheryl A Jones
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA
| | - Nicholas Pearce
- Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
| | - Carlie A Neiswanger
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA
| | - Min-Xuan Wang
- Universal Stabilization Technologies, Inc., San Diego, California, USA
| | - Erin A Miller
- Universal Stabilization Technologies, Inc., San Diego, California, USA
| | - Nikolai Petrovsky
- College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia
- Vaxine Pty Ltd, Warradale, South Australia, Australia
| | - David E Wentworth
- Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
| | - Victor Bronshtein
- Universal Stabilization Technologies, Inc., San Diego, California, USA
| | - Mark Papania
- Global Immunization Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
| | - Stephen M Tompkins
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA.
- Emory-UGA Centers of Excellence for Influenza Research and Surveillance (CEIRS), Athens, Georgia, 30602, USA.
- Department of Infectious Diseases, University of Georgia, Athens, Georgia, USA.
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13
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Tiwari D, Jakhmola S, Pathak DK, Kumar R, Jha HC. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein-Barr Virus Infection in Glial Cells. ACS OMEGA 2020; 5:29547-29560. [PMID: 33225186 PMCID: PMC7676301 DOI: 10.1021/acsomega.0c04525] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Accepted: 10/21/2020] [Indexed: 05/17/2023]
Abstract
Raman spectroscopy can be used as a tool to study virus entry and pathogen-driven manipulation of the host efficiently. To date, Epstein-Barr virus (EBV) entry and altered biochemistry of the glial cell upon infection are elusive. In this study, we detected biomolecular changes in human glial cells, namely, HMC-3 (microglia) and U-87 MG (astrocytes), at two variable cellular locations (nucleus and periphery) by Raman spectroscopy post-EBV infection at different time points. Two possible phenomena, one attributed to the response of the cell to viral attachment and invasion and the other involved in duplication of the virus followed by egress from the host cell, are investigated. These changes corresponded to unique Raman spectra associated with specific biomolecules in the infected and the uninfected cells. The Raman signals from the nucleus and periphery of the cell also varied, indicating differential biochemistry and signaling processes involved in infection progression at these locations. Molecules such as cholesterol, glucose, hyaluronan, phenylalanine, phosphoinositide, etc. are associated with the alterations in the cellular biochemical homeostasis. These molecules are mainly responsible for cellular processes such as lipid transport, cell proliferation, differentiation, and apoptosis in the cells. Raman signatures of these molecules at distinct time points of infection indicated their periodic involvement, depending on the stage of virus infection. Therefore, it is possible to discern the details of variability in EBV infection progression in glial cells at the biomolecular level using time-dependent in vitro Raman scattering.
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Affiliation(s)
- Deeksha Tiwari
- Discipline
of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol, 453552 Indore, India
| | - Shweta Jakhmola
- Discipline
of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol, 453552 Indore, India
| | - Devesh K. Pathak
- Discipline
of Physics, Indian Institute of Technology
Indore, Simrol, 453552 Indore, India
| | - Rajesh Kumar
- Discipline
of Physics, Indian Institute of Technology
Indore, Simrol, 453552 Indore, India
- Centre
for Advanced Electronics, Indian Institute
of Technology Indore, Simrol, 453552 Indore, India
| | - Hem Chandra Jha
- Discipline
of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol, 453552 Indore, India
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14
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Coupled CRC 2D and ALI 3D Cultures Express Receptors of Emerging Viruses and Are More Suitable for the Study of Viral Infections Compared to Conventional Cell Lines. Stem Cells Int 2020; 2020:2421689. [PMID: 32695180 PMCID: PMC7368225 DOI: 10.1155/2020/2421689] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2020] [Revised: 05/18/2020] [Accepted: 05/21/2020] [Indexed: 02/02/2023] Open
Abstract
Infections of emerging and reemerging viruses (SARS-CoVs, influenza H1N1, etc.) largely and globally affect human health. Animal models often fail to reflect a physiological status because of species tropism of virus infection. Conventional cell lines are usually genetically and phenotypically different from primary cells. Developing an in vitro physiological model to study the infection of emerging viruses will facilitate our understanding of virus-host cell interactions, thereby benefiting antiviral drug discovery. In the current work, we first established normal airway epithelial cells (upper and lower airway track) in 2D and 3D culture systems using conditional reprogramming (CR) and air-liquid interface (ALI) techniques. These long-term cultures maintained differentiation potential. More importantly, these cells express two types of influenza virus receptors, α2-6-Gal- and α2-3-Gal-linked sialic acids, and angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoVs as well. These cells were permissive to the infection of pandemic influenza H1N1 (H1N1pdm). In contrast, the lung cancer cell line A549 and immortalized airway epithelial cells (16HBE) were not susceptible to H1N1 infection. A virus-induced cytopathic effect (CPE) on 2D CRC cultures developed in a time-dependent manner. The pathological effects were also readily observed spreading from the apical layer to the basal layer of the 3D ALI culture. This integrated 2D CRC and 3D ALI cultures provide a physiological and personalized in vitro model to study the infection of emerging viruses. This novel model can be used for studying virus biology and host response to viral infection and for antiviral drug discovery.
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15
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Hawksworth A, Lockhart R, Crowe J, Maeso R, Ritter L, Dibben O, Bright H. Replication of live attenuated influenza vaccine viruses in human nasal epithelial cells is associated with H1N1 vaccine effectiveness. Vaccine 2020; 38:4209-4218. [PMID: 32376111 DOI: 10.1016/j.vaccine.2020.04.004] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2019] [Revised: 12/17/2019] [Accepted: 04/01/2020] [Indexed: 12/24/2022]
Abstract
In the 2013-2014 and 2015-2016 influenza seasons, live attenuated influenza vaccine (LAIV) generated reduced vaccine effectiveness (VE) against circulating H1N1 strains. This reduced VE coincided with the introduction of pandemic 2009 H1N1 (A/H1N1pdm09) vaccine virus reassortants, in place of pre-2009 seasonal H1N1 strains. Here, we explored one specific hypothesis for reduced VE; decreased replicative fitness of A/H1N1pdm09 strains in humans. Two A/H1N1pdm09 strains with reduced VE, A/California/07/2009 (A/CA09) and A/Bolivia/559/2013 (A/BOL13), were compared to pre-2009 seasonal H1N1 strains, A/New Caledonia/20/1999 (A/NC99) and A/South Dakota/6/2007 (A/SD07). Initial results showed that A/H1N1pdm09 strains had reduced multi-cycle infectivity in Madin-Darby Canine Kidney (MDCK) cells, compared to their pre-2009 counterparts. The A/BOL13 viral titre was found to be 2.65 log10/mL lower when measured by multi-cycle 50% tissue culture infectious dose (TCID50) assay compared to single-cycle fluorescent focus assay (FFA). By contrast, clinically effective A/NC99 titres differed by only 0.54 log10/mL. In human alveolar (A549) cells, A/H1N1pdm09 strains replicated less than pre-2009 strains, with A/CA09 and A/BOL13 generating lower peak viral titres over 5 days. This phenotype was corroborated in physiologically relevant, primary human nasal epithelial cells (hNECs). Here, peak titres for pre-2009 strains A/NC99 and A/SD07 were 8.43 log10 TCID50/mL and 8.52 log10 TCID50/mL, respectively, versus 6.89 log10 TCID50/mL and 6.06 log10 TCID50/mL for A/H1N1pdm09 strains A/CA09 and A/BOL13. This confirmed a reduced ability of A/H1N1pdm09 strains to sustain replication in human respiratory cells. Using this information, H1N1 candidate A/Slovenia/2903/2015 (A/SLOV15) was characterised for replacement of A/BOL13 in the 2017/18 LAIV. A/SLOV15 produced comparable single and multi-cycle infectivity titres (Δ 0.16 log10/mL) and reached a peak titre 1.23 log10 TCID50/mL higher than that of A/BOL13 in hNEC cultures. Taken together, these data suggest a reduction in sustained multi-cycle replication in human cells as a plausible root cause for reduced A/H1N1pdm09 VE.
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16
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Amouzougan EA, Lira R, Klimecki WT. Chronic exposure to arsenite enhances influenza virus infection in cultured cells. J Appl Toxicol 2020; 40:458-469. [PMID: 31960482 PMCID: PMC7931812 DOI: 10.1002/jat.3918] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 09/30/2019] [Accepted: 10/02/2019] [Indexed: 12/13/2022]
Abstract
Arsenic is a ubiquitous environmental toxicant that has been associated with human respiratory diseases. In humans, arsenic exposure has been associated with increased risk of respiratory infection. Considering the existing epidemiological evidence and the well-established impact of arsenic on epithelial cell biology, we posited that the effect of arsenic exposure in epithelial cells could enhance viral infection. In this study, we characterized influenza virus A/WSN/33 (H1N1) infection in Madin-Darby Canine Kidney (MDCK) cells chronically exposed to low levels of sodium arsenite (75 ppb). We observed a 27.3-fold increase in viral matrix (M2) protein (24 hours postinfection [p.i.]), a 1.35-fold increase in viral mRNA levels, and a 126% increase in plaque area in arsenite-exposed MDCK cells (48 hours p.i.). Arsenite exposure resulted in 114% increase in virus attachment-positive cells (2 hours p.i.) and 224% increase in α-2,3 sialic acid-positive cells. Interestingly, chronic exposure to arsenite reduced the effect of the antiviral drug, oseltamivir in MDCK cells. We also found that exposure to sodium arsenite resulted in a 4.4-fold increase in viral mRNA levels and significantly increased cytotoxicity in influenza A/Udorn/72 (H3N2) infected BEAS-2B cells. This study suggests that chronic arsenite exposure could result in enhanced influenza infection in epithelial cells, and that this may be mediated through increased sialic acid binding. Finally, the decreased effectiveness of the anti-influenza drug, oseltamivir, in arsenite-exposed cells raises substantial public health concerns if this effect translates to arsenic-exposed, influenza-infected people.
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Affiliation(s)
- Eva A. Amouzougan
- Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85724, United States
| | - Ricardo Lira
- Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85724, United States
| | - Walter T. Klimecki
- Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85724, United States
- College of Veterinary Medicine, The University of Arizona, Tucson, Arizona 85724, United States
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17
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Identification and Characterization of Novel Compounds with Broad-Spectrum Antiviral Activity against Influenza A and B Viruses. J Virol 2020; 94:JVI.02149-19. [PMID: 31941776 DOI: 10.1128/jvi.02149-19] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Accepted: 12/24/2019] [Indexed: 12/23/2022] Open
Abstract
Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC50) = 3.80 nM to 1.73 μM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC50 = 0.14 μM to 0.55 μM; SI-MTT = 70.12 to >357.14) or cotreatment (EC50 = 34.69 nM to 7.52 μM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections.IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.
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18
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Abstract
Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmission electron microscopy provides structural information, identity of components within a sample may be confounding. Immunoelectron microscopy allows for identification and visualization of antigens and their relative positions within a particulate sample. This allows for simple qualitative analysis of samples including whole virus, viral components, and viral‐like particles. This article describes methods for immunogold labeling of viral antigens in a liquid suspension, with examples of immunogold‐labeled influenza virus glycoproteins, and also discusses the important considerations for sample preparation and determination of morphologies. Together, these methods allow for understanding the antigenic makeup of viral particulate samples, which have important implications for molecular virology and vaccine development. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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Affiliation(s)
- Neetu M Gulati
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
| | - Udana Torian
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
| | - John R Gallagher
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
| | - Audray K Harris
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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19
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Wang Q, Bhattacharya S, Mereness JA, Anderson C, Lillis JA, Misra RS, Romas S, Huyck H, Howell A, Bandyopadhyay G, Donlon K, Myers JR, Ashton J, Pryhuber GS, Mariani TJ. A novel in vitro model of primary human pediatric lung epithelial cells. Pediatr Res 2020; 87:511-517. [PMID: 30776794 PMCID: PMC6698433 DOI: 10.1038/s41390-019-0340-9] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Revised: 01/07/2019] [Accepted: 02/04/2019] [Indexed: 12/05/2022]
Abstract
BACKGROUND Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. METHODS We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. RESULTS PHLE cells can be expanded in culture up to passage 6, with a doubling time of ~4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B, and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. CONCLUSION Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.
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Affiliation(s)
- Qian Wang
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
- Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA
- Center for Oral Biology, University of Rochester Medical Center, Rochester, NY, USA
| | - Soumyaroop Bhattacharya
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
- Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA
| | - Jared A Mereness
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
- Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA
- Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY, USA
| | - Christopher Anderson
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
- Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA
| | - Jacquelyn A Lillis
- Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY, USA
- UR Genomics Research Center, University of Rochester Medical Center, Rochester, NY, USA
| | - Ravi S Misra
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
| | - Stephen Romas
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
| | - Heidie Huyck
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
| | - Amanda Howell
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
| | - Gautam Bandyopadhyay
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
| | - Kathy Donlon
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
- Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA
| | - Jason R Myers
- UR Genomics Research Center, University of Rochester Medical Center, Rochester, NY, USA
| | - John Ashton
- UR Genomics Research Center, University of Rochester Medical Center, Rochester, NY, USA
| | - Gloria S Pryhuber
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA
| | - Thomas J Mariani
- Division of Neonatology, University of Rochester Medical Center, Rochester, NY, USA.
- Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA.
- Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY, USA.
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20
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Nelson SW, Lorbach JN, Nolting JM, Stull JW, Jackwood DJ, Davis IC, Bowman AS. Madin-Darby canine kidney cell sialic acid receptor modulation induced by culture medium conditions: Implications for the isolation of influenza A virus. Influenza Other Respir Viruses 2019; 13:593-602. [PMID: 31392833 PMCID: PMC6800301 DOI: 10.1111/irv.12671] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2018] [Revised: 06/25/2019] [Accepted: 06/28/2019] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND The influenza A virus (IAV) binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors expressed by Madin-Darby canine kidney (MDCK) cells. The receptor distribution may therefore be important in regulating IAV propagation. Serum-free medium (SFM) avoids variability in conventional culture medium containing fetal bovine serum (FBS), which can have variable composition and may contain endotoxins. However, little is known about the distribution of SA receptors on cells maintained in SFM. OBJECTIVES We assessed the influence of culture media on MDCK cell SA receptor distribution along with the effect of SA receptor distribution on IAV recovery. We hypothesized that SFM would increase the proportion of α-2,6-linked SA receptors present and alter isolate recovery. METHODS Madin-Darby canine kidney cells were cultured in medium containing FBS and two SFMs. Cell surface distribution of α-2,6- and α-2,3-linked receptors was determined using flow cytometry. Recovery of swine- and avian-lineage IAVs from MDCK cells maintained in each medium was quantified as TCID50 . RESULTS Madin-Darby canine kidney cells cultured in UltraMDCK SFM expressed both SA receptors and supported the growth of both swine- and avian-lineage IAVs. Cells maintained in other medium inconsistently expressed each receptor and the avian IAV grew to lower titers in cells cultured with FBS. CONCLUSIONS Medium conditions altered the distribution of SA receptors present on MDCK cells and affected IAV recovery. Culture in UltraMDCK SFM resulted in cells expressing both receptors and IAVs grew to higher titers than in the other culture condition, indicating that this medium may be useful for culturing IAV from multiple species.
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Affiliation(s)
- Sarah W. Nelson
- Veterinary Preventive MedicineThe Ohio State UniversityColumbusOhio
| | | | | | - Jason W. Stull
- Veterinary Preventive MedicineThe Ohio State UniversityColumbusOhio
| | - Daral J. Jackwood
- Veterinary Preventive MedicineThe Ohio State UniversityColumbusOhio
- Food Animal Health Research Program, The Ohio State UniversityWoosterOhio
| | - Ian C. Davis
- Veterinary Biosciences, The Ohio State UniversityColumbusOhio
| | - Andrew S. Bowman
- Veterinary Preventive MedicineThe Ohio State UniversityColumbusOhio
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21
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Chua SCJH, Tan HQ, Engelberg D, Lim LHK. Alternative Experimental Models for Studying Influenza Proteins, Host-Virus Interactions and Anti-Influenza Drugs. Pharmaceuticals (Basel) 2019; 12:E147. [PMID: 31575020 PMCID: PMC6958409 DOI: 10.3390/ph12040147] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 09/11/2019] [Accepted: 09/12/2019] [Indexed: 12/14/2022] Open
Abstract
Ninety years after the discovery of the virus causing the influenza disease, this malady remains one of the biggest public health threats to mankind. Currently available drugs and vaccines only partially reduce deaths and hospitalizations. Some of the reasons for this disturbing situation stem from the sophistication of the viral machinery, but another reason is the lack of a complete understanding of the molecular and physiological basis of viral infections and host-pathogen interactions. Even the functions of the influenza proteins, their mechanisms of action and interaction with host proteins have not been fully revealed. These questions have traditionally been studied in mammalian animal models, mainly ferrets and mice (as well as pigs and non-human primates) and in cell lines. Although obviously relevant as models to humans, these experimental systems are very complex and are not conveniently accessible to various genetic, molecular and biochemical approaches. The fact that influenza remains an unsolved problem, in combination with the limitations of the conventional experimental models, motivated increasing attempts to use the power of other models, such as low eukaryotes, including invertebrate, and primary cell cultures. In this review, we summarized the efforts to study influenza in yeast, Drosophila, zebrafish and primary human tissue cultures and the major contributions these studies have made toward a better understanding of the disease. We feel that these models are still under-utilized and we highlight the unique potential each model has for better comprehending virus-host interactions and viral protein function.
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Affiliation(s)
- Sonja C J H Chua
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore.
- NUS Immunology Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
- CREATE-NUS-HUJ Molecular Mechanisms of Inflammatory Diseases Programme, National University of Singapore, Singapore 138602, Singapore.
| | - Hui Qing Tan
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore.
- NUS Immunology Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
| | - David Engelberg
- CREATE-NUS-HUJ Molecular Mechanisms of Inflammatory Diseases Programme, National University of Singapore, Singapore 138602, Singapore.
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore.
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
| | - Lina H K Lim
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore.
- NUS Immunology Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
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22
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Pepin KM, Hopken MW, Shriner SA, Spackman E, Abdo Z, Parrish C, Riley S, Lloyd-Smith JO, Piaggio AJ. Improving risk assessment of the emergence of novel influenza A viruses by incorporating environmental surveillance. Philos Trans R Soc Lond B Biol Sci 2019; 374:20180346. [PMID: 31401963 PMCID: PMC6711309 DOI: 10.1098/rstb.2018.0346] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Reassortment is an evolutionary mechanism by which influenza A viruses (IAV) generate genetic novelty. Reassortment is an important driver of host jumps and is widespread according to retrospective surveillance studies. However, predicting the epidemiological risk of reassortant emergence in novel hosts from surveillance data remains challenging. IAV strains persist and co-occur in the environment, promoting co-infection during environmental transmission. These conditions offer opportunity to understand reassortant emergence in reservoir and spillover hosts. Specifically, environmental RNA could provide rich information for understanding the evolutionary ecology of segmented viruses, and transform our ability to quantify epidemiological risk to spillover hosts. However, significant challenges with recovering and interpreting genomic RNA from the environment have impeded progress towards predicting reassortant emergence from environmental surveillance data. We discuss how the fields of genomics, experimental ecology and epidemiological modelling are well positioned to address these challenges. Coupling quantitative disease models and natural transmission studies with new molecular technologies, such as deep-mutational scanning and single-virus sequencing of environmental samples, should dramatically improve our understanding of viral co-occurrence and reassortment. We define observable risk metrics for emerging molecular technologies and propose a conceptual research framework for improving accuracy and efficiency of risk prediction. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.
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Affiliation(s)
- Kim M. Pepin
- National Wildlife Research Center, USDA-APHIS, Fort Collins, CO 80521, USA
- e-mail:
| | - Matthew W. Hopken
- National Wildlife Research Center, USDA-APHIS, Fort Collins, CO 80521, USA
- Colorado State University, Fort Collins, CO 80523, USA
| | - Susan A. Shriner
- National Wildlife Research Center, USDA-APHIS, Fort Collins, CO 80521, USA
| | - Erica Spackman
- Exotic and Emerging Avian Viral Diseases Research, USDA-ARS, Athens, GA 30605, USA
| | - Zaid Abdo
- Colorado State University, Fort Collins, CO 80523, USA
| | - Colin Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853, USA
| | - Steven Riley
- MRC Centre for Global Infectious Disease Analysis, Imperial College, London, SW7 2AZ, UK
| | - James O. Lloyd-Smith
- UCLA, Los Angeles, CA 90095, USA
- Department of Ecology and Evolutionary Biology, Fogarty International Center, National Institutes of Health, Bethesda MD 20892, USA
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23
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Lindsey BB, Jagne YJ, Armitage EP, Singanayagam A, Sallah HJ, Drammeh S, Senghore E, Mohammed NI, Jeffries D, Höschler K, Tregoning JS, Meijer A, Clarke E, Dong T, Barclay W, Kampmann B, de Silva TI. Effect of a Russian-backbone live-attenuated influenza vaccine with an updated pandemic H1N1 strain on shedding and immunogenicity among children in The Gambia: an open-label, observational, phase 4 study. THE LANCET. RESPIRATORY MEDICINE 2019; 7:665-676. [PMID: 31235405 PMCID: PMC6650545 DOI: 10.1016/s2213-2600(19)30086-4] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/11/2019] [Revised: 03/03/2019] [Accepted: 03/04/2019] [Indexed: 01/19/2023]
Abstract
BACKGROUND The efficacy and effectiveness of the pandemic H1N1 (pH1N1) component in live attenuated influenza vaccine (LAIV) is poor. The reasons for this paucity are unclear but could be due to impaired replicative fitness of pH1N1 A/California/07/2009-like (Cal09) strains. We assessed whether an updated pH1N1 strain in the Russian-backbone trivalent LAIV resulted in greater shedding and immunogenicity compared with LAIV with Cal09. METHODS We did an open-label, prospective, observational, phase 4 study in Sukuta, a periurban area in The Gambia. We enrolled children aged 24-59 months who were clinically well. Children received one dose of the WHO prequalified Russian-backbone trivalent LAIV containing either A/17/California/2009/38 (Cal09) or A/17/New York/15/5364 (NY15) based on their year of enrolment. Primary outcomes were the percentage of children with LAIV strain shedding at day 2 and day 7, haemagglutinin inhibition seroconversion, and an increase in influenza haemagglutinin-specific IgA and T-cell responses at day 21 after LAIV. This study is nested within a randomised controlled trial investigating LAIV-microbiome interactions (NCT02972957). FINDINGS Between Feb 8, 2017, and April 12, 2017, 118 children were enrolled and received one dose of the Cal09 LAIV from 2016-17. Between Jan 15, 2018, and March 28, 2018, a separate cohort of 135 children were enrolled and received one dose of the NY15 LAIV from 2017-18, of whom 126 children completed the study. Cal09 showed impaired pH1N1 nasopharyngeal shedding (16 of 118 children [14%, 95% CI 8·0-21·1] with shedding at day 2 after administration of LAIV) compared with H3N2 (54 of 118 [46%, 36·6-55·2]; p<0·0001) and influenza B (95 of 118 [81%, 72·2-87·2]; p<0·0001), along with suboptimal serum antibody (seroconversion in six of 118 [5%, 1·9-10·7]) and T-cell responses (CD4+ interferon γ-positive and/or CD4+ interleukin 2-positive responses in 45 of 111 [41%, 31·3-50·3]). After the switch to NY15, a significant increase in pH1N1 shedding was seen (80 of 126 children [63%, 95% CI 54·4-71·9]; p<0·0001 compared with Cal09), along with improvements in seroconversion (24 of 126 [19%, 13·2-26·8]; p=0·011) and influenza-specific CD4+ T-cell responses (73 of 111 [66%, 60·0-75·6; p=0·00028]). The improvement in pH1N1 seroconversion with NY15 was even greater in children who were seronegative at baseline (24 of 64 children [38%, 95% CI 26·7-49·8] vs six of 79 children with Cal09 [8%, 2·8-15·8]; p<0·0001). Persistent shedding to day 7 was independently associated with both seroconversion (odds ratio 12·69, 95% CI 4·1-43·6; p<0·0001) and CD4+ T-cell responses (odds ratio 7·83, 95% CI 2·99-23·5; p<0·0001) by multivariable logistic regression. INTERPRETATION The pH1N1 component switch that took place between 2016 and 2018 might have overcome the poor efficacy and effectiveness reported with previous LAIV formulations. LAIV effectiveness against pH1N1 should, therefore, improve in upcoming influenza seasons. Our data highlight the importance of assessing replicative fitness, in addition to antigenicity, when selecting annual LAIV components. FUNDING The Wellcome Trust.
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Affiliation(s)
- Benjamin B Lindsey
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia; Department of Medicine, Imperial College London, London, UK
| | - Ya Jankey Jagne
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - Edwin P Armitage
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | | | - Hadijatou J Sallah
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - Sainabou Drammeh
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - Elina Senghore
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - Nuredin I Mohammed
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - David Jeffries
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - Katja Höschler
- Virus Reference Department, Reference Microbiology Services, Public Health England, London, UK
| | | | - Adam Meijer
- Centre for Infectious Disease Research, Diagnostics and Laboratory Surveillance, National Institute for Public Health and the Environment, Bilthoven, Netherlands
| | - Ed Clarke
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia
| | - Tao Dong
- Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, and Chinese Academy of Medical Science-Oxford Institute, Nuffield Department of Medicine, Oxford University, Oxford, UK
| | - Wendy Barclay
- Department of Medicine, Imperial College London, London, UK
| | - Beate Kampmann
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia; The Vaccine Centre, London School of Hygiene & Tropical Medicine, Faculty of Infectious and Tropical Diseases, London, UK
| | - Thushan I de Silva
- Vaccines and Immunity Theme, Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia; Department of Medicine, Imperial College London, London, UK; The Florey Institute for Host-Pathogen Interactions and Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK.
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24
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Michalak P, Soszynska-Jozwiak M, Biala E, Moss WN, Kesy J, Szutkowska B, Lenartowicz E, Kierzek R, Kierzek E. Secondary structure of the segment 5 genomic RNA of influenza A virus and its application for designing antisense oligonucleotides. Sci Rep 2019; 9:3801. [PMID: 30846846 PMCID: PMC6406010 DOI: 10.1038/s41598-019-40443-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2018] [Accepted: 02/12/2019] [Indexed: 12/20/2022] Open
Abstract
Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (-)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics.
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Affiliation(s)
- Paula Michalak
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Marta Soszynska-Jozwiak
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Ewa Biala
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Walter N Moss
- Roy J. Carver Department of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, 50011, USA
| | - Julita Kesy
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Barbara Szutkowska
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Elzbieta Lenartowicz
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Ryszard Kierzek
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland
| | - Elzbieta Kierzek
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland.
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25
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Czakó R, Vogel L, Sutton T, Matsuoka Y, Krammer F, Chen Z, Jin H, Subbarao K. H5N2 vaccine viruses on Russian and US live attenuated influenza virus backbones demonstrate similar infectivity, immunogenicity and protection in ferrets. Vaccine 2018; 36:1871-1879. [PMID: 29503113 PMCID: PMC5854182 DOI: 10.1016/j.vaccine.2018.02.061] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Revised: 02/09/2018] [Accepted: 02/15/2018] [Indexed: 11/19/2022]
Abstract
The continued detection of zoonotic influenza infections, most notably due to the avian influenza A H5N1 and H7N9 subtypes, underscores the need for pandemic preparedness. Decades of experience with live attenuated influenza vaccines (LAIVs) for the control of seasonal influenza support the safety and effectiveness of this vaccine platform. All LAIV candidates are derived from one of two licensed master donor viruses (MDVs), cold-adapted (ca) A/Ann Arbor/6/60 or ca A/Leningrad/134/17/57. A number of LAIV candidates targeting avian H5 influenza viruses derived with each MDV have been evaluated in humans, but have differed in their infectivity and immunogenicity. To understand these differences, we generated four H5N2 candidate pandemic LAIVs (pLAIVs) derived from either MDV and compared their biological characteristics in vitro and in vivo. We demonstrate that all candidate pLAIVs, regardless of gene constellation and derivation, were comparable with respect to infectivity, immunogenicity, and protection from challenge in the ferret model of influenza. These observations suggest that differences in clinical performance of H5 pLAIVs may be due to factors other than inherent biological properties of the two MDVs.
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Affiliation(s)
- Rita Czakó
- Emerging Respiratory Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, USA
| | - Leatrice Vogel
- Emerging Respiratory Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, USA
| | - Troy Sutton
- Emerging Respiratory Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, USA
| | - Yumiko Matsuoka
- Emerging Respiratory Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, USA
| | - Florian Krammer
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | | | - Hong Jin
- MedImmune Vaccines, Mountain View, CA, USA
| | - Kanta Subbarao
- Emerging Respiratory Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, USA.
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26
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Soszynska-Jozwiak M, Michalak P, Moss WN, Kierzek R, Kesy J, Kierzek E. Influenza virus segment 5 (+)RNA - secondary structure and new targets for antiviral strategies. Sci Rep 2017; 7:15041. [PMID: 29118447 PMCID: PMC5678188 DOI: 10.1038/s41598-017-15317-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2017] [Accepted: 10/24/2017] [Indexed: 01/05/2023] Open
Abstract
Influenza A virus is a threat for humans due to seasonal epidemics and occasional pandemics. This virus can generate new strains that are dangerous through nucleotide/amino acid changes or through segmental recombination of the viral RNA genome. It is important to gain wider knowledge about influenza virus RNA to create new strategies for drugs that will inhibit its spread. Here, we present the experimentally determined secondary structure of the influenza segment 5 (+)RNA. Two RNAs were studied: the full-length segment 5 (+)RNA and a shorter construct containing only the coding region. Chemical mapping data combined with thermodynamic energy minimization were used in secondary structure prediction. Sequence/structure analysis showed that the determined secondary structure of segment 5 (+)RNA is mostly conserved between influenza virus type A strains. Microarray mapping and RNase H cleavage identified accessible sites for oligonucleotides in the revealed secondary structure of segment 5 (+)RNA. Antisense oligonucleotides were designed based on the secondary structure model and tested against influenza virus in cell culture. Inhibition of influenza virus proliferation was noticed, identifying good targets for antisense strategies. Effective target sites fall within two domains, which are conserved in sequence/structure indicating their importance to the virus.
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Affiliation(s)
- Marta Soszynska-Jozwiak
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704, Poznan, Noskowskiego 12/14, Poland
| | - Paula Michalak
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704, Poznan, Noskowskiego 12/14, Poland
| | - Walter N Moss
- Roy J. Carver Department of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, 50011, United States of America
| | - Ryszard Kierzek
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704, Poznan, Noskowskiego 12/14, Poland
| | - Julita Kesy
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704, Poznan, Noskowskiego 12/14, Poland
| | - Elzbieta Kierzek
- Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704, Poznan, Noskowskiego 12/14, Poland.
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27
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Wohlgemuth N, Ye Y, Fenstermacher KJ, Liu H, Lane AP, Pekosz A. The M2 protein of live, attenuated influenza vaccine encodes a mutation that reduces replication in human nasal epithelial cells. Vaccine 2017; 35:6691-6699. [PMID: 29079099 DOI: 10.1016/j.vaccine.2017.10.018] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2017] [Revised: 10/05/2017] [Accepted: 10/08/2017] [Indexed: 12/31/2022]
Abstract
The influenza A virus components of the live, attenuated influenza vaccine (LAIV) encode the HA and NA gene segments from a circulating virus strain and the remaining gene segments from the cold-adapted master donor virus, A/Ann Arbor/6/1960 (H2N2). The master donor virus imparts at least three phenotypes: temperature-sensitivity (ts), attenuation (att), and cold-adaption (ca). The genetic loci responsible for the att and ts phenotypes of LAIV were mapped to PB1, PB2, and NP by reverse genetics experiments using immortalized cell lines. However, some in vivo studies have demonstrated that the M segment, which acquired an alanine (Ala) to serine (Ser) mutation at M2 position 86 during cold adaption - a mutation found in no other influenza A virus strain - contributes to the att phenotype. Prior studies have shown this region of the M2 cytoplasmic tail to be critical for influenza virus replication. Using reverse genetics, we demonstrate that certain amino acid substitutions at M2 positions 83 and 86 alter the replication of influenza A/Udorn/307/72 (H3N2). Importantly, substitution of a Ser at M2 position 86 reduces A/Udorn/307/72 replication in differentiated primary human nasal epithelial cell (hNECs) cultures, but does not considerably affect replication in MDCK cells. When a Ser was substituted for Ala at M2 86 in LAIV, the virus replicated to higher titers and with faster kinetics in hNEC cultures, implicating this amino acid change as contributing to LAIV attenuation. Increased replication also resulted in increased production of IFN-λ. These data indicate the LAIV associated Ser mutation at M2 position 86 contributes to the att phenotype and is associated with a differential regulation of interferon in LAIV infection.
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Affiliation(s)
- Nicholas Wohlgemuth
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Yang Ye
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Katherine J Fenstermacher
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Hsuan Liu
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Andrew P Lane
- Department of Otolaryngology - Head and Neck Surgery, Johns Hopkins Outpatient Center, Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Andrew Pekosz
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
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28
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Forero A, Fenstermacher K, Wohlgemuth N, Nishida A, Carter V, Smith EA, Peng X, Hayes M, Francis D, Treanor J, Morrison J, Klein SL, Lane A, Katze MG, Pekosz A. Evaluation of the innate immune responses to influenza and live-attenuated influenza vaccine infection in primary differentiated human nasal epithelial cells. Vaccine 2017; 35:6112-6121. [PMID: 28967519 DOI: 10.1016/j.vaccine.2017.09.058] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2016] [Revised: 09/13/2017] [Accepted: 09/19/2017] [Indexed: 01/08/2023]
Abstract
The host innate immune response to influenza virus is a key determinant of pathogenic outcomes and long-term protective immune responses against subsequent exposures. Here, we present a direct contrast of the host responses in primary differentiated human nasal epithelial cell (hNEC) cultures following infection with either a seasonal H3N2 influenza virus (WT) or the antigenically-matched live-attenuated vaccine (LAIV) strain. Comparison of the transcriptional profiles obtained 24 and 36h post-infection showed that the magnitude of gene expression was greater in LAIV infected relative to that observed in WT infected hNEC cultures. Functional enrichment analysis revealed that the antiviral and inflammatory responses were largely driven by type III IFN induction in both WT and LAIV infected cells. However, the enrichment of biological pathways involved in the recruitment of mononuclear leukocytes, antigen-presenting cells, and T lymphocytes was uniquely observed in LAIV infected cells. These observations were reflective of the host innate immune responses observed in individuals acutely infected with influenza viruses. These findings indicate that cell-intrinsic type III IFN-mediated innate immune responses in the nasal epithelium are not only crucial for viral clearance and attenuation, but may also play an important role in the induction of protective immune responses with live-attenuated vaccines.
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Affiliation(s)
- Adriana Forero
- Department of Microbiology, University of Washington, Seattle, WA, USA
| | - Katherine Fenstermacher
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA
| | - Nicholas Wohlgemuth
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA
| | - Andrew Nishida
- Department of Microbiology, University of Washington, Seattle, WA, USA
| | - Victoria Carter
- Department of Microbiology, University of Washington, Seattle, WA, USA
| | - Elise A Smith
- Department of Microbiology, University of Washington, Seattle, WA, USA
| | - Xinxia Peng
- Department of Microbiology, University of Washington, Seattle, WA, USA
| | - Melissa Hayes
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA
| | - Doreen Francis
- Department of Internal Medicine, University of Rochester, School of Medicine and Dentistry, Rochester, New York, USA
| | - John Treanor
- Department of Internal Medicine, University of Rochester, School of Medicine and Dentistry, Rochester, New York, USA
| | - Juliet Morrison
- Department of Microbiology, University of Washington, Seattle, WA, USA
| | - Sabra L Klein
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA
| | - Andrew Lane
- Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, School of Medicine, Baltimore, MD, USA
| | - Michael G Katze
- Department of Microbiology, University of Washington, Seattle, WA, USA; Washington National Primate Research Center, University of Washington, Seattle, WA, USA
| | - Andrew Pekosz
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA; Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA.
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Singanayagam A, Zambon M, Lalvani A, Barclay W. Urgent challenges in implementing live attenuated influenza vaccine. THE LANCET. INFECTIOUS DISEASES 2017; 18:e25-e32. [PMID: 28780285 DOI: 10.1016/s1473-3099(17)30360-2] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Revised: 05/10/2017] [Accepted: 05/25/2017] [Indexed: 12/26/2022]
Abstract
Conflicting reports have emerged about the effectiveness of the live attenuated influenza vaccine. The live attenuated influenza vaccine appears to protect particularly poorly against currently circulating H1N1 viruses that are derived from the 2009 pandemic H1N1 viruses. During the 2015-16 influenza season, when pandemic H1N1 was the predominant virus, studies from the USA reported a complete lack of effectiveness of the live vaccine in children. This finding led to a crucial decision in the USA to recommend that the live vaccine not be used in 2016-17 and to switch to the inactivated influenza vaccine. Other countries, including the UK, Canada, and Finland, however, have continued to recommend the use of the live vaccine. This policy divergence and uncertainty has far reaching implications for the entire global community, given the importance of the production capabilities of the live attenuated influenza vaccine for pandemic preparedness. In this Personal View, we discuss possible explanations for the observed reduced effectiveness of the live attenuated influenza vaccine and highlight the underpinning scientific questions. Further research to understand the reasons for these observations is essential to enable informed public health policy and commercial decisions about vaccine production and development in coming years.
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Affiliation(s)
- Anika Singanayagam
- Department of Medicine, Imperial College, London, UK; NIHR Health Protection Research Unit in Respiratory Infections, Imperial College, London, UK
| | - Maria Zambon
- Virus Reference Department, National Infection Service, Public Health England, Colindale, London, UK; NIHR Health Protection Research Unit in Respiratory Infections, Imperial College, London, UK
| | - Ajit Lalvani
- National Heart and Lung Institute, Imperial College, London, UK; NIHR Health Protection Research Unit in Respiratory Infections, Imperial College, London, UK
| | - Wendy Barclay
- Department of Medicine, Imperial College, London, UK; NIHR Health Protection Research Unit in Respiratory Infections, Imperial College, London, UK.
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Establishment of MDCK/FX Cell for Efficient Replication of Influenza Viruses. Jundishapur J Microbiol 2017. [DOI: 10.5812/jjm.44891] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
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31
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Kumar A, Kim JH, Ranjan P, Metcalfe MG, Cao W, Mishina M, Gangappa S, Guo Z, Boyden ES, Zaki S, York I, García-Sastre A, Shaw M, Sambhara S. Influenza virus exploits tunneling nanotubes for cell-to-cell spread. Sci Rep 2017; 7:40360. [PMID: 28059146 PMCID: PMC5216422 DOI: 10.1038/srep40360] [Citation(s) in RCA: 114] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2015] [Accepted: 12/06/2016] [Indexed: 02/06/2023] Open
Abstract
Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naïve cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naïve cell, resulting in productive viral replication in the naïve cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures.
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Affiliation(s)
- Amrita Kumar
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Jin Hyang Kim
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Priya Ranjan
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Maureen G Metcalfe
- Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Weiping Cao
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Margarita Mishina
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Shivaprakash Gangappa
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Zhu Guo
- Virus Surveillance and Diagnostics Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Edward S Boyden
- Media Lab, McGovern Institute, Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA
| | - Sherif Zaki
- Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Ian York
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Adolfo García-Sastre
- Department of Microbiology, Department of Infectious Disease, Global Health and Emerging Pathogens Institute and Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, USA
| | - Michael Shaw
- Office of Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
| | - Suryaprakash Sambhara
- Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA
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Guido M, Tumolo MR, Verri T, Romano A, Serio F, De Giorgi M, De Donno A, Bagordo F, Zizza A. Human bocavirus: Current knowledge and future challenges. World J Gastroenterol 2016; 22:8684-8697. [PMID: 27818586 PMCID: PMC5075545 DOI: 10.3748/wjg.v22.i39.8684] [Citation(s) in RCA: 116] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2016] [Revised: 08/30/2016] [Accepted: 09/14/2016] [Indexed: 02/06/2023] Open
Abstract
Human bocavirus (HBoV) is a parvovirus isolated about a decade ago and found worldwide in both respiratory samples, mainly from early life and children of 6-24 mo of age with acute respiratory infection, and in stool samples, from patients with gastroenteritis. Since then, other viruses related to the first HBoV isolate (HBoV1), namely HBoV2, HBoV3 and HBoV4, have been detected principally in human faeces. HBoVs are small non-enveloped single-stranded DNA viruses of about 5300 nucleotides, consisting of three open reading frames encoding the first two the non-structural protein 1 (NS1) and nuclear phosphoprotein (NP1) and the third the viral capsid proteins 1 and 2 (VP1 and VP2). HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models for the difficulties in replicating the virus in in vitro cell cultures, and the fact that HBoV infection is frequently accompanied by at least another viral and/or bacterial respiratory and/or gastroenteric pathogen infection. Current diagnostic methods to support HBoV detection include polymerase chain reaction, real-time PCR, enzyme-linked immunosorbent assay and enzyme immunoassay using recombinant VP2 or virus-like particle capsid proteins, although sequence-independent amplification techniques combined with next-generation sequencing platforms promise rapid and simultaneous detection of the pathogens in the future. This review presents the current knowledge on HBoV genotypes with emphasis on taxonomy, phylogenetic relationship and genomic analysis, biology, epidemiology, pathogenesis and diagnostic methods. The emerging discussion on HBoVs as true pathogen or innocent bystander is also emphasized.
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Sonnberg S, Ducatez MF, DeBeauchamp J, Crumpton JC, Rubrum A, Sharp B, Hall RJ, Peacey M, Huang S, Webby RJ. Pandemic Seasonal H1N1 Reassortants Recovered from Patient Material Display a Phenotype Similar to That of the Seasonal Parent. J Virol 2016; 90:7647-56. [PMID: 27279619 PMCID: PMC4988147 DOI: 10.1128/jvi.00772-16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2016] [Accepted: 06/06/2016] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED We have previously shown that 11 patients became naturally coinfected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during coinfection of host animals and in vitro; however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two coinfecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally coinfected patients. IMPORTANCE Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural coinfection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.
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Affiliation(s)
| | | | | | | | - Adam Rubrum
- St. Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Bridgett Sharp
- St. Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Richard J Hall
- Institute of Environmental Science and Research, Upper Hutt, New Zealand
| | - Matthew Peacey
- Institute of Environmental Science and Research, Upper Hutt, New Zealand
| | - Sue Huang
- Institute of Environmental Science and Research, Upper Hutt, New Zealand
| | - Richard J Webby
- St. Jude Children's Research Hospital, Memphis, Tennessee, USA
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McWhite CD, Meyer AG, Wilke CO. Sequence amplification via cell passaging creates spurious signals of positive adaptation in influenza virus H3N2 hemagglutinin. Virus Evol 2016; 2:vew026. [PMID: 27713835 PMCID: PMC5049878 DOI: 10.1093/ve/vew026] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Clinical influenza A virus isolates are frequently not sequenced directly. Instead, a majority of these isolates (~70% in 2015) are first subjected to passaging for amplification, most commonly in non-human cell culture. Here, we find that this passaging leaves distinct signals of adaptation, which can confound evolutionary analyses of the viral sequences. We find distinct patterns of adaptation to Madin-Darby (MDCK) and monkey cell culture absent from unpassaged hemagglutinin sequences. These patterns also dominate pooled datasets not separated by passaging type, and they increase in proportion to the number of passages performed. By contrast, MDCK-SIAT1 passaged sequences seem mostly (but not entirely) free of passaging adaptations. Contrary to previous studies, we find that using only internal branches of influenza virus phylogenetic trees is insufficient to correct for passaging artifacts. These artifacts can only be safely avoided by excluding passaged sequences entirely from subsequent analysis. We conclude that future influenza virus evolutionary analyses should appropriately control for potentially confounding effects of passaging adaptations.
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Affiliation(s)
- Claire D. McWhite
- Center for Systems and Synthetic Biology and Institute for Cellular and
Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA
- Department of Molecular Biosciences, The University of Texas at Austin,
Austin, TX 78712, USA
| | - Austin G. Meyer
- Center for Systems and Synthetic Biology and Institute for Cellular and
Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA
- Center for Computational Biology and Bioinformatics, The University of Texas
at Austin, Austin, TX 78712, USA
- Department of Integrative Biology, The University of Texas at Austin,
Austin, TX 78712, USA
| | - Claus O. Wilke
- Center for Systems and Synthetic Biology and Institute for Cellular and
Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA
- Center for Computational Biology and Bioinformatics, The University of Texas
at Austin, Austin, TX 78712, USA
- Department of Integrative Biology, The University of Texas at Austin,
Austin, TX 78712, USA
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35
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Ilyushina NA, Wright PF. In vitro modeling of the interaction between human epithelial cells and lymphocytes upon influenza infection. Influenza Other Respir Viruses 2016; 10:438-42. [PMID: 27102577 PMCID: PMC4947944 DOI: 10.1111/irv.12394] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/12/2016] [Indexed: 12/01/2022] Open
Abstract
Influenza viruses are a continuous threat to humans because of their ability to cross species barriers and adapt to new hosts. Data from murine studies, along with limited human data, suggest that CD8+ cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural influenza proteins are the main mediators of influenza virus clearance. Additionally, the fact that many CTLs recognize epitopes shared between different influenza strains offers the potential for broad cross‐strain immunity. However, the mechanisms of cellular immunity against influenza viruses are poorly defined in humans, where the CTL response has been hard to measure and interpret. We developed a novel CTL assay that utilizes fully differentiated nasal human epithelial cells taken from volunteers as permissive targets for autologous peripheral blood‐derived influenza virus‐specific cytotoxic T lymphocytes. This in vitro system of human lymphocyte–epithelial cell co‐cultures can be considered as the closest approximation to events in vivo and can be employed for studying the interactions between the pathogen and human host. Modeling of the natural interaction process between the primary cell type that supports the productive replication of influenza and immune cells may allow us to put in perspective CTLs as a correlate of immunity to influenza in humans.
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Affiliation(s)
- Natalia A Ilyushina
- Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA
| | - Peter F Wright
- Department of Pediatrics, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
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Shanmuganatham KK, Jones JC, Marathe BM, Feeroz MM, Jones-Engel L, Walker D, Turner J, Rabiul Alam SM, Kamrul Hasan M, Akhtar S, Seiler P, McKenzie P, Krauss S, Webby RJ, Webster RG. The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals. Emerg Microbes Infect 2016; 5:e35. [PMID: 27094903 PMCID: PMC4855072 DOI: 10.1038/emi.2016.29] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2015] [Revised: 12/29/2015] [Accepted: 01/06/2016] [Indexed: 01/28/2023]
Abstract
H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.
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Affiliation(s)
| | - Jeremy C Jones
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Bindumadhav M Marathe
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Mohammed M Feeroz
- Department of Zoology, Jahangirnagar University, Dhaka 1342, Bangladesh
| | - Lisa Jones-Engel
- National Primate Research Center University of Washington, Seattle, WA 98195-5502, USA
| | - David Walker
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Jasmine Turner
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - S M Rabiul Alam
- Department of Zoology, Jahangirnagar University, Dhaka 1342, Bangladesh
| | - M Kamrul Hasan
- Department of Zoology, Jahangirnagar University, Dhaka 1342, Bangladesh
| | - Sharmin Akhtar
- Department of Zoology, Jahangirnagar University, Dhaka 1342, Bangladesh
| | - Patrick Seiler
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Pamela McKenzie
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Scott Krauss
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Richard J Webby
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Robert G Webster
- Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA
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Khatri M, Chattha KS. Replication of influenza A virus in swine umbilical cord epithelial stem-like cells. Virulence 2016; 6:40-9. [PMID: 25517546 DOI: 10.4161/21505594.2014.983020] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
In this study, we describe the isolation and characterization of epithelial stem-like cells from the swine umbilical cord and their susceptibility to influenza virus infection. Swine umbilical cord epithelial stem cells (SUCECs) expressed stem cell and pluripotency associated markers such as SSEA-1, SSEA-4, TRA 1-60 and TRA 1-81 and Oct4. Morphologically, cells displayed polygonal morphology and were found to express epithelial markers; pancytokeratin, cytokeratin-18 and occludin; mesenchymal cell markers CD44, CD90 and haematopoietic cell marker CD45 were not detected on these cells. The cells had extensive proliferation and self- renewal properties. The cells also possessed immunomodulatory activity and inhibited the proliferation of T cells. Also, higher levels of anti-inflammatory cytokine IL-10 were detected in SUCEC-T cell co-cultures. The cells were multipotent and differentiated into lung epithelial cells when cultured in epithelial differentiation media. We also examined if SUCECs are susceptible to infection with influenza virus. SUCECs expressed sialic acid receptors, used by influenza virus for binding to cells. The 2009 pandemic influenza virus and swine influenza virus replicated in these cells. SUCECs due to their differentiation and immunoregulatory properties will be useful as cellular therapy in a pig model for human diseases. Additionally, our data indicate that influenza virus can infect SUCECs and may transmit influenza virus from mother to fetus through umbilical cord and transplantation of influenza virus-infected stem cells may transmit infection to recipients. Therefore, we propose that umbilical cord cells, in addition to other agents, should also be tested for influenza virus before cryopreservation for future use as a cell therapy for disease conditions.
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Affiliation(s)
- Mahesh Khatri
- a Food Animal Health Research Program; Ohio Agricultural Research and Development Center; The Ohio State University ; Wooster , OH United States
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Role of Substitutions in the Hemagglutinin in the Emergence of the 1968 Pandemic Influenza Virus. J Virol 2015; 89:12211-6. [PMID: 26378170 DOI: 10.1128/jvi.01292-15] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 09/02/2015] [Indexed: 01/03/2023] Open
Abstract
Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differs from the putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for adaptation of avian HA to mammals. We found that introduction of avian-virus-like amino acids at five other HA positions (positions 62, 81, 92, 144, and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.
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Isakova-Sivak I, Rudenko L. Safety, immunogenicity and infectivity of new live attenuated influenza vaccines. Expert Rev Vaccines 2015; 14:1313-29. [PMID: 26289975 DOI: 10.1586/14760584.2015.1075883] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Live attenuated influenza vaccines (LAIVs) are believed to be immunologically superior to inactivated influenza vaccines, because they can induce a variety of adaptive immune responses, including serum antibodies, mucosal and cell-mediated immunity. In addition to the licensed cold-adapted LAIV backbones, a number of alternative LAIV approaches are currently being developed and evaluated in preclinical and clinical studies. This review summarizes recent progress in the development and evaluation of LAIVs, with special attention to their safety, immunogenicity and infectivity for humans, and discusses their perspectives for the future.
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Affiliation(s)
- Irina Isakova-Sivak
- a Department of Virology, Institute of Experimental Medicine, 12 Acad. Pavlov Street, Saint Petersburg, Russia
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40
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Restricted replication of the live attenuated influenza A virus vaccine during infection of primary differentiated human nasal epithelial cells. Vaccine 2015. [PMID: 26196325 DOI: 10.1016/j.vaccine.2015.07.023] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Live Attenuated Influenza Vaccine (LAIV) strains are associated with cold adapted, temperature sensitive and attenuated phenotypes that have been studied in non-human or immortalized cell cultures as well as in animal models. Using a primary, differentiated human nasal epithelial cell (hNEC) culture system we compared the replication kinetics, levels of cell-associated viral proteins and virus particle release during infection with LAIV or the corresponding wild type (WT) influenza viruses. At both 33 °C and 37 °C, seasonal influenza virus and an antigenically matched LAIV replicated to similar titers in MDCK cells but seasonal influenza virus replicated to higher titers than LAIV in hNEC cultures, suggesting a greater restriction of LAIV replication in hNEC cultures. Despite the disparity in infectious virus production, the supernatants from H1N1 and LAIV infected hNEC cultures had equivalent amounts of viral proteins and hemagglutination titers, suggesting the formation of non-infectious virus particles by LAIV in hNEC cultures.
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Davis AS, Chertow DS, Moyer JE, Suzich J, Sandouk A, Dorward DW, Logun C, Shelhamer JH, Taubenberger JK. Validation of normal human bronchial epithelial cells as a model for influenza A infections in human distal trachea. J Histochem Cytochem 2015; 63:312-28. [PMID: 25604814 PMCID: PMC4409941 DOI: 10.1369/0022155415570968] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2014] [Accepted: 01/05/2015] [Indexed: 11/22/2022] Open
Abstract
Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.
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Affiliation(s)
- A Sally Davis
- Viral Pathogenesis and Evolution Section, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland (ASD, DSC, JEM, AS, JKT)
- Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine, Manhattan, Kansas (ASD)
| | - Daniel S Chertow
- Viral Pathogenesis and Evolution Section, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland (ASD, DSC, JEM, AS, JKT)
- Critical Care Medicine Department, Clinical Center, NIH, Bethesda, Maryland (DSC, JS, CL, JHS)
| | - Jenna E Moyer
- Viral Pathogenesis and Evolution Section, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland (ASD, DSC, JEM, AS, JKT)
| | - Jon Suzich
- Critical Care Medicine Department, Clinical Center, NIH, Bethesda, Maryland (DSC, JS, CL, JHS)
| | - Aline Sandouk
- Viral Pathogenesis and Evolution Section, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland (ASD, DSC, JEM, AS, JKT)
| | - David W Dorward
- Electron Microscopy Unit, Research Technology Branch, NIAID, Hamilton, Montana (DWD)
| | - Carolea Logun
- Critical Care Medicine Department, Clinical Center, NIH, Bethesda, Maryland (DSC, JS, CL, JHS)
| | - James H Shelhamer
- Critical Care Medicine Department, Clinical Center, NIH, Bethesda, Maryland (DSC, JS, CL, JHS)
| | - Jeffery K Taubenberger
- Viral Pathogenesis and Evolution Section, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland (ASD, DSC, JEM, AS, JKT)
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Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus. Arch Virol 2015; 160:1397-405. [DOI: 10.1007/s00705-015-2395-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2014] [Accepted: 03/08/2015] [Indexed: 12/14/2022]
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Daidoji T, Watanabe Y, Ibrahim MS, Yasugi M, Maruyama H, Masuda T, Arai F, Ohba T, Honda A, Ikuta K, Nakaya T. Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH. J Biol Chem 2015; 290:10627-42. [PMID: 25673693 DOI: 10.1074/jbc.m114.611327] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2014] [Indexed: 12/13/2022] Open
Abstract
The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells.
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Affiliation(s)
- Tomo Daidoji
- From the Department of Infectious Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
| | - Yohei Watanabe
- the Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
| | - Madiha S Ibrahim
- the Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan, the Department of Microbiology, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22111, Egypt
| | - Mayo Yasugi
- the Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan, the Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, 598-8531, Japan
| | - Hisataka Maruyama
- the Department of Micro-Nano Systems Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, and
| | - Taisuke Masuda
- the Department of Micro-Nano Systems Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, and
| | - Fumihito Arai
- the Department of Micro-Nano Systems Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, and
| | - Tomoyuki Ohba
- the Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, Japan
| | - Ayae Honda
- the Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, Japan
| | - Kazuyoshi Ikuta
- the Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
| | - Takaaki Nakaya
- From the Department of Infectious Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan,
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High basal expression of interferon-stimulated genes in human bronchial epithelial (BEAS-2B) cells contributes to influenza A virus resistance. PLoS One 2014; 9:e109023. [PMID: 25313647 PMCID: PMC4196766 DOI: 10.1371/journal.pone.0109023] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2014] [Accepted: 08/27/2014] [Indexed: 11/19/2022] Open
Abstract
Respiratory epithelial cells play a key role in influenza A virus (IAV) pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV-host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77) IAVs in normal primary human bronchial epithelial (NHBE) and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK) cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7), stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK) inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo.
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Strengert M, Jennings R, Davanture S, Hayes P, Gabriel G, Knaus UG. Mucosal reactive oxygen species are required for antiviral response: role of Duox in influenza a virus infection. Antioxid Redox Signal 2014; 20:2695-709. [PMID: 24128054 DOI: 10.1089/ars.2013.5353] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
AIMS Influenza A virus (IAV), a major airborne pathogen, is closely associated with significant morbidity and mortality. The primary target for influenza virus replication is the respiratory epithelium, which reacts to infection by mounting a multifaceted antiviral response. A part of this mucosal host defense is the generation of reactive oxygen species (ROS) by NADPH oxidases. Duox1 and Duox2 are the main ROS-producing enzymes in the airway epithelium, but their contribution to mammalian host defense is still ill defined. RESULTS To gain a better understanding of Duox function in respiratory tract infections, human differentiated lung epithelial cells and an animal model were used to monitor the effect of epithelial ROS on IAV propagation. IAV infection led to coordinated up-regulation of Duox2 and Duox-mediated ROS generation. Interference with H2O2 production and ROS signaling by oxidase inhibition or H2O2 decomposition augmented IAV replication. A nuclear pool of Duox enzymes participated in the regulation of the spliceosome, which is critical for alternative splicing of viral transcripts and controls the assembly of viable virions. In vivo silencing of Duox increased the viral load on intranasal infection with 2009 pandemic H1N1 influenza virus. INNOVATION This is the first study conclusively linking Duox NADPH oxidases with the antiviral mammalian immune response. Further, ROS generated by Duox enzymes localized adjacent to nuclear speckles altered the splicing of viral genes. CONCLUSION Duox-derived ROS are host protective and essential for counteracting IAV replication.
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Fischer WA, Chason KD, Brighton M, Jaspers I. Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures. Vaccine 2014; 32:1761-7. [PMID: 24486351 DOI: 10.1016/j.vaccine.2013.12.069] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2013] [Revised: 12/08/2013] [Accepted: 12/24/2013] [Indexed: 01/27/2023]
Abstract
Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.
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Affiliation(s)
- William A Fischer
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA; The Center for Environmental Medicine, Asthma and Lung Biology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
| | - Kelly D Chason
- Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, 8033 Burnett-Womack, Chapel Hill, NC 27599-7219, USA
| | - Missy Brighton
- The Center for Environmental Medicine, Asthma and Lung Biology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Ilona Jaspers
- The Center for Environmental Medicine, Asthma and Lung Biology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA; Department of Pediatrics, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
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Jones JC, Baranovich T, Marathe BM, Danner AF, Seiler JP, Franks J, Govorkova EA, Krauss S, Webster RG. Risk assessment of H2N2 influenza viruses from the avian reservoir. J Virol 2014; 88:1175-88. [PMID: 24227848 PMCID: PMC3911670 DOI: 10.1128/jvi.02526-13] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Accepted: 10/24/2013] [Indexed: 11/20/2022] Open
Abstract
H2N2 influenza A viruses were the cause of the 1957-1958 pandemic. Historical evidence demonstrates they arose from avian virus ancestors, and while the H2N2 subtype has disappeared from humans, it persists in wild and domestic birds. Reemergence of H2N2 in humans is a significant threat due to the absence of humoral immunity in individuals under the age of 50. Thus, examination of these viruses, particularly those from the avian reservoir, must be addressed through surveillance, characterization, and antiviral testing. The data presented here are a risk assessment of 22 avian H2N2 viruses isolated from wild and domestic birds over 6 decades. Our data show that they have a low rate of genetic and antigenic evolution and remained similar to isolates circulating near the time of the pandemic. Most isolates replicated in mice and human bronchial epithelial cells, but replication in swine tissues was low or absent. Multiple isolates replicated in ferrets, and 3 viruses were transmitted to direct-contact cage mates. Markers of mammalian adaptation in hemagglutinin (HA) and PB2 proteins were absent from all isolates, and they retained a preference for avian-like α2,3-linked sialic acid receptors. Most isolates remained antigenically similar to pandemic A/Singapore/1/57 (H2N2) virus, suggesting they could be controlled by the pandemic vaccine candidate. All viruses were susceptible to neuraminidase inhibitors and adamantanes. Nonetheless, the sustained pathogenicity of avian H2N2 viruses in multiple mammalian models elevates their risk potential for human infections and stresses the need for continual surveillance as a component of prepandemic planning.
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Affiliation(s)
- Jeremy C Jones
- Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
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Lugovtsev VY, Melnyk D, Weir JP. Heterogeneity of the MDCK cell line and its applicability for influenza virus research. PLoS One 2013; 8:e75014. [PMID: 24058646 PMCID: PMC3772841 DOI: 10.1371/journal.pone.0075014] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2013] [Accepted: 08/08/2013] [Indexed: 11/18/2022] Open
Abstract
Single-cell clones have been established from the MDCK cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Three discrete cell morphotypes were identified using light microscopy. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin (PNA). All clones were susceptible to infection by influenza viruses of different subtypes of influenza A virus (H1N1, H1N1pdm09, H3N2, H5N1) and influenza B virus, and all possessed on their surface terminally sialylated glycans with both types of glycosidic linkage (α2-3 and α2-6). The Type-1 cell lines were able to support a multicycle replication of influenza A and B viruses without help of an exogenous trypsin. In contrast, cell lines exhibiting Type-2 morphology were unable to support multicycle replication of influenza A viruses without trypsin supplementation. Western blot analysis of the hemagglutinin of H1N1 strains demonstrated that Type-2 cells were deficient in production of proteolytically activated hemagglutinin (no cleavage between HA1/HA2 was observed). HA1/HA2 cleavage of influenza B viruses in the Type-2 cells was also significantly impaired, but not completely abrogated, producing sufficient amount of activated HA to support efficient virus replication without trypsin. In contrast, all clones of Type-1 cells were able to produce proteolytically activated hemagglutinin of influenza A and B viruses. However, the growth kinetics and plaque size of influenza A viruses varied significantly in different clones. Influenza B virus also showed different plaque size, with the biggest plaque formation in the Type-2 cells, although the growth kinetics and peak infectivity titers were similar in all clones. Taken together, the study demonstrates that the population of original MDCK cells is represented by various types of cells that differ in their capacities to support replication of influenza A and B viruses.
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Affiliation(s)
- Vladimir Y. Lugovtsev
- Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America
- * E-mail:
| | - Darya Melnyk
- Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America
| | - Jerry P. Weir
- Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America
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Teng Z, Kuang X, Wang J, Zhang X. Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity. J Virol Methods 2013; 193:364-70. [PMID: 23835032 DOI: 10.1016/j.jviromet.2013.06.034] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2013] [Revised: 06/19/2013] [Accepted: 06/26/2013] [Indexed: 11/19/2022]
Abstract
A newly developed electronic cell sensor array--the xCELLigence real-time cell analysis (RTCA) system is tested currently for dynamic monitoring of cell attachment, proliferation, damage, and death. In this study, human enterovirus (HEV71) infection of human rhabdomyosarcoma (RD) was used as an in vitro model to validate the application of this novel system as a straightforward and efficient assay for quantitative measurement of infectious viruses based on virus-induced cytopathic effect (CPE). Several experimental tests were performed including the determination of optimal seeding density of the RD cells in 96-well E-plates, RTCA real-time monitoring of the virus induced CPE and virus titer calculation, and viral neutralization test to determine HEV71 antibody titer. Traditional 50% tissue culture infective dose (TCID50) assay was also conducted for methodology comparison and validation, which indicated a consistent result between the two assays. These findings indicate that the xCELLigence RTCA system can be a valuable addition to current viral assays for quantitative measurement of infectious viruses and quantitation of neutralization antibody titer in real-time, warranting for future research and exploration of applications to many other animal and human viruses.
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Affiliation(s)
- Zheng Teng
- Department of Microbiology, Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China
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50
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Abstract
Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.
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