scholarly journals Establishment of a genetically engineered chicken DF-1 cell line for efficient amplification of influenza viruses in the absence of trypsin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kelly Chungu ◽  
Young Hyun Park ◽  
Seung Je Woo ◽  
Su Bin Lee ◽  
Deivendran Rengaraj ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Cell Lines ◽  
Influenza Infection ◽  
Initial Step ◽  
Genetically Engineered ◽  
Influenza Viruses ◽  
Host Cells ◽  
Viral Titer ◽  
Chicken Cell

Abstract Background The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. In cell culture systems, HA is cleaved by trypsin added to the culture medium. The vast majority of established cell lines are mammalian. Results In the present study, we generated genetically engineered chicken DF-1 cell lines overexpressing transmembrane protease, serine 2 (TMPRSS2, which cleaves HA), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1, which plays a role in synthesis of α-2,3 linked sialic acids to which avian-adapted viruses bind preferentially), or both. We found that overexpression of TMPRSS2 supports the virus life cycle by cleaving HA. Furthermore, we found that overexpression of ST3GAL1 increased the viral titer. Finally, we showed that overexpression of both TMPRSS2 and ST3GAL1 increased the final viral titer due to enhanced support of viral replication and prolonged viability of the cells. In addition, overexpression of these genes of interest had no effect on cell proliferation and viability. Conclusions Taken together, the results indicate that these engineered cells could be used as a cell-based system to propagate influenza virus efficiently in the absence of trypsin. Further studies on influenza virus interactions with chicken cell host factors could be studied without the effect of trypsin on cells.

scholarly journals Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
Author(s):  
Daniel R. Perez ◽  
Ruben O. Donis
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Virus Growth ◽  
Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

scholarly journals Knockout of IRF7 Highlights its Modulator Function of Host Response Against Avian Influenza Virus and the Involvement of MAPK and TOR Signaling Pathways in Chicken

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 385
Author(s):  
Tae Hyun Kim ◽  
Colin Kern ◽  
Huaijun Zhou
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Host Response ◽  
Antiviral Response ◽  
Influenza Viruses ◽  
Interferon Response ◽  
Type I

Interferon regulatory factor 7 (IRF7) is known as the master transcription factor of the type I interferon response in mammalian species along with IRF3. Yet birds only have IRF7, while they are missing IRF3, with a smaller repertoire of immune-related genes, which leads to a distinctive immune response in chickens compared to in mammals. In order to understand the functional role of IRF7 in the regulation of the antiviral response against avian influenza virus in chickens, we generated IRF7-/- chicken embryonic fibroblast (DF-1) cell lines and respective controls (IRF7wt) by utilizing the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. IRF7 knockout resulted in increased viral titers of low pathogenic avian influenza viruses. Further RNA-sequencing performed on H6N2-infected IRF7-/- and IRF7wt cell lines revealed that the deletion of IRF7 resulted in the significant down-regulation of antiviral effectors and the differential expression of genes in the MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) signaling pathways. Dynamic gene expression profiling of the host response between the wildtype and IRF7 knockout revealed potential signaling pathways involving AP1 (activator protein 1), NF-κB (nuclear factor kappa B) and inflammatory cytokines that may complement chicken IRF7. Our findings in this study provide novel insights that have not been reported previously, and lay a solid foundation for enhancing our understanding of the host antiviral response against the avian influenza virus in chickens.

scholarly journals RNA-Sequencing-Based Transcriptomic Analysis Reveals a Role for Annexin-A1 in Classical and Influenza A Virus-Induced Autophagy

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1399 ◽  
Author(s):  
Jianzhou Cui ◽  
Dhakshayini Morgan ◽  
Dao Han Cheng ◽  
Sok Lin Foo ◽  
Gracemary L. R. Yap ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Infection ◽  
Critical Role ◽  
Mtor Inhibitors ◽  
Influenza Viruses ◽  
Annexin A1 ◽  
Underlying Mechanisms

Influenza viruses have been shown to use autophagy for their survival. However, the proteins and mechanisms involved in the autophagic process triggered by the influenza virus are unclear. Annexin-A1 (ANXA1) is an immunomodulatory protein involved in the regulation of the immune response and Influenza A virus (IAV) replication. In this study, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated protein 9) deletion of ANXA1, combined with the next-generation sequencing, we systematically analyzed the critical role of ANXA1 in IAV infection as well as the detailed processes governing IAV infection, such as macroautophagy. A number of differentially expressed genes were uniquely expressed in influenza A virus-infected A549 parental cells and A549 ∆ANXA1 cells, which were enriched in the immune system and infection-related pathways. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the role of ANXA1 in autophagy. To validate this, the effect of mechanistic target of rapamycin (mTOR) inhibitors, starvation and influenza infection on autophagy was determined, and our results demonstrate that ANXA1 enhances autophagy induced by conventional autophagy inducers and influenza virus. These results will help us to understand the underlying mechanisms of IAV infection and provide a potential therapeutic target for restricting influenza viral replication and infection.

scholarly journals Discovery of Allopregnanolone Against Influenza Virus With Broad Spectrum in Vitro

2021 ◽  
Author(s):  
yuqi Wang ◽  
Yanyan Wang ◽  
Hong Cao
Keyword(s):  
Natural Products ◽  
Influenza Virus ◽  
Viral Replication ◽  
Broad Spectrum ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
Dependent Manner ◽  
Viral Yield

Abstract Background: Influenza virus infection with seasonal or occasional but devastating morbidity and mortality, is a severe threat to public health. The frequent emergence of resistant viral strains limited application of current antivirals and posing an urgent need for novel antiviral therapies. Natural products offered a broad prospect in the screening and development of new influenza inhibitors.Methods: In this research, a high-throughput antiviral screening for 891 natural products was performed based on a recombinant reporter influenza A virus. According to the cytotoxicity assay and dose-response relationship, alloprogesterone (ALLO), as the positive hit was selected, and verified by viral titer reduction assay and immunofluorescence using a wild-type virus. Followingly, we explored its antiviral potency of counteracting with IAV and IBV, and preliminary investigated the mechanism of ALLO through time-of-addition assay and mini-replicon system.Results: Under the criteria of 80% inhibition and 70% cell viability, ALLO was screened out and confirmed antiviral activity in varied cells. The inhibitory effect of ALLO against influenza virus with a dose-dependent manner and significantly reduced viral yield of five different influenza viruses in the presence of 40 µM ALLO, including oseltamivir-resistant virus. Moreover, ALLO exhibited no influence on IAV entry or release during the viral replication cycle, but obviously interfered with the genome replication regarding post-infection 2 hrs to 6 hrs, which is consistent with the evidence of decreased polymerase activity.Conclusions: In summary, we firstly identified a new pharmacological activity of ALLO, as a broad spectrum inhibitor for treatment influenza infections, targeting viral replication stage and possessing great value of further development.

scholarly journals Influenza virus inhibits respiratory syncytial virus (RSV) infection via a two-wave expression of interferon-induced protein with tetratricopeptide (IFIT) proteins

2020 ◽  
Author(s):  
Yaron Drori ◽  
Jasmine Jacob-Hirsch ◽  
Rakefet Pando ◽  
Aharona Glatman-Freedman ◽  
Nehemya Friedman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Influenza Virus ◽  
Respiratory Syncytial Virus ◽  
Influenza Infection ◽  
Treatment Strategies ◽  
Respiratory Viruses ◽  
Influenza Viruses ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Mouse Lungs

AbstractInfluenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance shows that RSV infection generally precedes influenza. However, in the last four winter seasons (2016-2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza virus inhibits RSV we performed coinfection of Human cervical carcinoma (HEp2) cells or mice with influenza and RSV and we observed that the influenza inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with tetratricopeptide (IFITs) family. Interestingly, in the second peak of upregulation, influenza viruses were no longer detectable in mouse lungs. We also observed that knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding of the interaction between influenza and RSV viruses and immune system involvement will contribute to the development and optimization of future treatment strategies against these viruses.Author SummaryRespiratory syncytial virus (RSV) and influenza viruses are both respiratory viruses associated with morbidity and mortality worldwide. RSV is usually detected in October, with a clear peak in December, whereas influenza virus arrives in November and peaks in January. In the last four seasons, influenza infection overlapped with that of RSV in Israel, which resulted in decreased morbidity of RSV suggesting that influenza virus inhibits RSV infection. To identify the mechanism responsible for the influenza inhibition of RSV we performed experiments in culture and in mice. We observed that influenza infection results in two wave modality of inhibition of RSV infection. Using mass spectrometry perfornmed on lungs from infected mice we show that influenza infection induces the expression of (IFIT) family of proteins which also showed a two-wave modality. Using knockdown and overexpression experiments we showed that indeed the IFTIs inhibits RSV infection. Our study provides new insights on the interaction between influenza and RSV viruses and immune system involvement and contribute to the development of future treatment strategies against these viruses.

scholarly journals Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Victoria Meliopoulos ◽  
Sean Cherry ◽  
Nicholas Wohlgemuth ◽  
Rebekah Honce ◽  
Karen Barnard ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Influenza A ◽  
Vaccine Development ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Clinical Samples ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial

ABSTRACT Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

The Oncolytic Herpes Simplex Virus Talimogene Laherparepvec Shows Promising Efficacy in Neuroendocrine Cancer Cell Lines

2019 ◽  
Vol 109 (4) ◽  
pp. 346-361 ◽  
Author(s):  
Linus D. Kloker ◽  
Susanne Berchtold ◽  
Irina Smirnow ◽  
Martin Schaller ◽  
Birgit Fehrenbacher ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Cell Lines ◽  
Genetically Engineered ◽  
Talimogene Laherparepvec ◽  
Electron Microscopic ◽  
Therapy Regimen ◽  
Neuroendocrine Cancer ◽  
Simplex Virus

Metastatic neuroendocrine cancer still constitutes a palliative situation, lacking promising treatment options. Oncolytic virotherapy, a novel type of virus-based immunotherapy, lyses tumor cells using genetically engineered viruses thereby activating the immune system to induce an optimized antitumor response which could bring down tumor masses to a stage of minimal residual tumor disease. The oncolytic vector talimogene laherparepvec (T-VEC, herpes simplex virus [HSV] type 1) has already shown excellent safety profiles in clinical studies and has become the first ever FDA/EMA-approved oncolytic virus (OV). This work presents a first preclinical assessment of this state-of-the-art OV, using a panel of human neuroendocrine tumor/neuroendocrine carcinoma (NET/NEC) cell lines. Cytotoxicity, transgene expression, and viral replication patterns were studied. Furthermore, the antiproliferative activity was compared to the one of mTOR inhibitor Everolimus and also interactions between the OV and Everolimus were evaluated. Moreover, virostatic effects of ganciclovir (GCV) on replication of T-VEC were assessed and electron microscopic pictures were taken to comprehend viral envelopment and details of the replication cycle of T-VEC in human neuroendocrine cancer. It could be shown that T-VEC infects, replicates in, and lyses human NET/NEC cells exhibiting high oncolytic efficiencies already at quite low virus concentrations. Interestingly, Everolimus was not found to have any relevant impact on rates of viral replication, but no additive effects could be proved using a combinatorial therapy regimen. On the other hand, GCV was shown to be able to limit replication of T-VEC, thus establishing an important safety feature for future treatments of NET/NEC patients. Taken together, T-VEC opens up a promising novel treatment option for NET/NEC patients, warranting its further preclinical and clinical development.

scholarly journals High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 462
Author(s):  
Jeffrey W. Ecker ◽  
Greg A. Kirchenbaum ◽  
Spencer R. Pierce ◽  
Amanda L. Skarlupka ◽  
Rodrigo B. Abreu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Influenza Viruses ◽  
Scale Production ◽  
Vaccine Platform ◽  
Mammalian Cell Lines

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1949 ◽  
Vol 90 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Chick Embryo ◽  
Active Agent ◽  
Allantoic Fluid ◽  
Inhibitory Effect ◽  
Influenza Viruses ◽  
Host Cells ◽  
Active Virus ◽  
Homologous Virus ◽  
Host Virus Interactions

Experiments have been reported on the propagation of influenza viruses in the allantoic membrane of the developing chick embryo during the first infectious cycle. After adsorption of the seed virus onto the host cells, only a small percentage of it remains demonstrable by infectivity titrations. This amount remains constant for 4 hours in the case of infection with PR8 virus, and for 6 hours in that of infection with Lee virus. Thereafter, a sharp rise in infectivity occurs 2 to 3 hours before liberation of the new generations of active virus into the allantoic fluid can be detected. Injection of homologous virus, inactivated by ultraviolet irradiation, following infection prevents or delays the production of virus in the tissues, depending to some extent upon the number of ID50 of active virus used as inoculum. The smaller the dose, the more pronounced the inhibitory effect. With increasing delay in the injection of the inhibitor, progressively more virus is produced and liberated 6 and 9 hours after infection with PR8 and Lee virus, respectively. Thus, production of virus may be interrupted by the homologous inhibitor when given up to 3 hours after infection with PR8, and up to4½ hours after infection with Lee virus. Since no increase in infectivity can bedetected during these 3 and 4½ hour periods in the tissues, it is suggested that influenza virus propagates in at least two major stages: first, non-infectious, immature virus material is produced which, subsequently, is converted into the fully active agent. Presumably the first step can be interrupted by the homologous inhibitor, while the second cannot. Heterologous irradiated virus, injected after infection of the tissue, exerts only a slight inhibitory effect on the production of virus.

scholarly journals Sialic Acid-Binding ProteinSp2CBMTD Protects Mice against Lethal Challenge with Emerging Influenza A (H7N9) Virus

2014 ◽  
Vol 59 (3) ◽  
pp. 1495-1504 ◽  
Author(s):  
Elena A. Govorkova ◽  
Tatiana Baranovich ◽  
Bindumadhav M. Marathe ◽  
Lei Yang ◽  
Margaret A. Taylor ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Influenza A ◽  
Adaptive Immune Response ◽  
Genetically Engineered ◽  
Influenza Viruses ◽  
H7n9 Virus ◽  
Lethal Challenge ◽  
Proinflammatory Mediators

ABSTRACTCompounds that target the cellular factors essential for influenza virus replication represent an innovative approach to antiviral therapy.Sp2CBMTD is a genetically engineered multivalent protein that masks sialic acid-containing cellular receptors on the respiratory epithelium, which are recognized by influenza viruses. Here, we evaluated the antiviral potential ofSp2CBMTD against lethal infection in mice with an emerging A/Anhui/1/2013 (H7N9) influenza virus and addressed the mechanistic basis of its activityin vivo. Sp2CBMTD was administered to mice intranasally as a single or repeated dose (0.1, 1, 10, or 100 μg) before (day −7, −3, and/or −1) or after (6 or 24 h) H7N9 virus inoculation. A singleSp2CBMTD dose (10 or 100 μg) protected 80% to 100% of the mice when administered 7 days before the H7N9 lethal challenge. RepeatedSp2CBMTD administration conferred the highest protection, resulting in 100% survival of the mice even at the lowest dose tested (0.1 μg). When treatment began 24 h after exposure to the H7N9 virus, a single administration of 100 μg ofSp2CBMTD protected 40% of the mice from death. The administration ofSp2CBMTD induced the pulmonary expression of proinflammatory mediators (interleukin-6 [IL-6], IL-1β, RANTES, monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein-1α [MIP-1α], and inducible protein [IP-10]) and recruited neutrophils to the respiratory tract before H7N9 virus infection, which resulted in less pronounced inflammation and rapid virus clearance from mouse lungs.Sp2CBMTD administration did not affect the virus-specific adaptive immune response, which was sufficient to protect against reinfection with a higher dose of homologous H7N9 virus or heterologous H5N1 virus. Thus,Sp2CBMTD was effective in preventing H7N9 infections in a lethal mouse model and holds promise as a prophylaxis option against zoonotic influenza viruses.

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