Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

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Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18189525 ◽

2021 ◽

Vol 18 (18) ◽

pp. 9525

Author(s):

Maria Antonia De Francesco ◽

Caterina Pollara ◽

Franco Gargiulo ◽

Mauro Giacomelli ◽

Arnaldo Caruso

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Statistical Significance ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Contact Tracing ◽

Nucleic Acid Detection ◽

Respiratory Pathogens ◽

Syncytial Virus ◽

Human Coronaviruses

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017–February 2020 (pre-pandemic period) and March 2020–May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

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

10.1101/2020.08.17.253708 ◽

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.

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Generation of chimeric respiratory viruses with altered tropism by coinfection of influenza A virus and respiratory syncytial virus

10.1101/2021.08.16.456460 ◽

2021 ◽

Author(s):

Joanne Haney ◽

Swetha Vijayakrishnan ◽

James Streetley ◽

Kieran Dee ◽

Daniel Max Goldfarb ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A Virus ◽

Influenza A ◽

Electron Tomography ◽

Super Resolution ◽

Respiratory Viruses ◽

Cellular Level ◽

Human Lung Cells ◽

Syncytial Virus ◽

Virus Interactions

SummaryInteractions between co-circulating respiratory viruses are recognized for their impact on viral transmission and clinical outcomes. However, the consequences of these virus-virus interactions at the cellular level are unclear. We coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy combined with live-cell imaging and scanning electron microscopy identified extracellular and membrane-associated filamentous structures, likely composed of elements of both IAV and RSV virions. Cryo-electron tomography confirmed the presence of chimeric virus particles exhibiting glycoproteins and ribonucleoproteins of both parental viruses. Functional assays revealed chimeric particles facilitate IAV infection in cells depleted of IAV receptors, demonstrating expanded tropism. Our observations define a previously unknown interaction that is likely to affect virus pathogenesis and have profound implications for infection biology.

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Differential Type I Interferon Induction by Respiratory Syncytial Virus and Influenza A Virus In Vivo

Journal of Virology ◽

10.1128/jvi.00530-07 ◽

2007 ◽

Vol 81 (18) ◽

pp. 9790-9800 ◽

Cited By ~ 113

Author(s):

Nancy A. Jewell ◽

Negin Vaghefi ◽

Sara E. Mertz ◽

Parvis Akter ◽

R. Stokes Peebles ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Type I Interferon ◽

Type I ◽

Type I Ifn ◽

Syncytial Virus

ABSTRACTType I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-α/β induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-α/β production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-α/β in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.

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Development of a Rapid Automated Influenza A, Influenza B, and Respiratory Syncytial Virus A/B Multiplex Real-Time RT-PCR Assay and Its Use during the 2009 H1N1 Swine-Origin Influenza Virus Epidemic in Milwaukee, Wisconsin

Journal of Molecular Diagnostics ◽

10.2353/jmoldx.2010.090095 ◽

2010 ◽

Vol 12 (1) ◽

pp. 74-81 ◽

Cited By ~ 27

Author(s):

Eric T. Beck ◽

Lisa A. Jurgens ◽

Sue C. Kehl ◽

Michael E. Bose ◽

Teresa Patitucci ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Real Time ◽

Influenza A ◽

Influenza B ◽

Rt Pcr ◽

Pcr Assay ◽

2009 H1n1 ◽

Syncytial Virus

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DETECTION OF INFLUENZA VIRUS AND PATHOGENS OF ACUTE RESPIRATORY VIRAL INFECTIONS IN POPULATION OF KAZAKHSTAN DURING 2018-2019 EPIDEMIC SEASON

Russian Journal of Infection and Immunity ◽

10.15789/2220-7619-doi-1348 ◽

2019 ◽

Author(s):

N. G. Klivleyeva ◽

N. S. Ongarbayeva ◽

A. M. Baimukhametova ◽

N. T. Saktaganov ◽

G. V. Lukmanova ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Influenza A ◽

Viral Infections ◽

Influenza Viruses ◽

Clinical Samples ◽

Type B ◽

Epidemic Season ◽

Parainfluenza Viruses ◽

Syncytial Virus

Influenza and other acute respiratory viral infections are the most common contemporary infectious diseases resulting in prominent harm to human health and great economic damage. At least five groups of viruses including more than 300 subtypes are currently referred to ARVI pathogens. Such infectious agents are characterized by variability resulting in their altered antigenic characteristics, increased contagiousness, "evasion from immune response and resistance to antivirals. Relevance of influenza and other ARVIs is also accounted for by rapid development of bacteria-associated respiratory diseases. Continuous variability of influenza viruses and emergence of new ARVI pathogens pose a serious threat. In recent years, a simultaneous circulation of subtype A (H1N1) and A (H3N2) influenza viruses with a predominance of a pandemic strain as well as type B viruses have been observed. Among the causative agents of non-influenza ARVIs, respiratory syncytial virus, rhino- and adenoviruses, and I/III parainfluenza viruses are recorded most often. Here we present the data of virology and serological examination of clinical samples collected during the 2018 – 2019 epidemic season in the Republic of Kazakhstan. For this, 2794 clinical samples (2530 nasopharyngeal swabs and 264 blood serums) were collected from patients diagnosed with ARVI, ARI, bronchitis, and pneumonia. Analysis of nasopharyngeal swabs for detection of influenza by RT-PCR demonstrated that mixed etiology influenza viruses with predominance of A/H1N1pdm virus circulated in Kazakhstan. The genetic fingerprints of influenza virus were found in 511 swabs (20.20% of total examined samples). Influenza A virus RNA was detected in 508 biological samples: A/H1N1 – in 289, A/H3N2 – in 209, and unidentified virus subtype in 10 samples. Type B influenza virus was detected in 3 samples. Study of 264 serum samples by HAI assay and ELISA showed emergence of antibodies against influenza A/H1N1, A/H3N2, and B viruses in residents from various regions of Kazakhstan that indirectly confirmed co-circulation of these viruses. 42 influenza virus strains were isolated in chicken embryos, from which 28 were assigned to A/H1N1pdm virus, 13 to A/H3N2 virus, and one isolate was identified as influenza B virus. Laboratory diagnostics of clinical samples for ARVIs established that among identified non-influenza agents respiratory syncytial virus dominated, while rhinoviruses and adenoviruses were less common. Metapneumoviruses, bocaviruses, coronaviruses, and type I parainfluenza viruses were detected in few cases. Comparison of study data with those obtained after examining circulation of influenza viruses during the 2017 – 2018 epidemic season showed that in 2018 – 2019 in Kazakhstan similar to the previous epidemic season, influenza A and B viruses continued to circulate, with prevalence of A/H1N1pdm virus. Identification of non-influenza viruses causing respiratory infections in 2018 – 2019 showed predominance of respiratory syncytial virus, which correlated with data on the 2017 – 2018 epidemic season.

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Discovery of a novel specific inhibitor targeting influenza A nucleoprotein with pleiotropic inhibitory effects on various steps of viral life cycle

Journal of Virology ◽

10.1128/jvi.01432-20 ◽

2021 ◽

Author(s):

Fang Yang ◽

Bo Pang ◽

Kin Kui Lai ◽

Nam Nam Cheung ◽

Jun Dai ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Broad Spectrum ◽

Influenza A ◽

Recurrent Events ◽

Antiviral Drug ◽

Influenza Virus Infection ◽

Specific Inhibitor ◽

Influenza Viruses ◽

Novel Influenza A

Influenza viruses (IAV) continue to pose an imminent threat to human due to annual influenza epidemics outbreak and episodic pandemics with high mortality. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for new additional prophylactic and therapeutic options, including new antiviral targets and drugs with new mechanisms of action to prevent and treat influenza infection. Here we characterized a novel influenza A nucleoprotein (NP) inhibitor FA-6005 that inhibited a broad spectrum of human pandemic, seasonal influenza A and B viruses in vitro and protects mice against lethal influenza A virus challenge. The small molecule FA-6005 targeted a conserved NP I41 domain and acted as a potential broad, multi-mechanistic anti-influenza virus therapeutic since FA-6005 suppressed influenza virus replication and perturbed intracellular trafficking of viral ribonucleoproteins (vRNP) from early to late stage. Cocrystal structures of the NP/FA-6005 complex reconciled well with concurrent mutational studies. This study provides the first line of direct evidence suggesting that the newly-identified NP I41 pocket as an attractive target for drug development that inhibit the multiple functions of NP. Our results also highlighted FA-6005 as a promising candidate for further development as an antiviral drug for the treatment of IAV infection and provide chemical-level details for inhibitor optimization. Importance Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Therefore, there is an urgent need for broad-spectrum inhibitors to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and the emergence of antiviral drug resistance. Herein we identified a novel influenza A virus NP antagonist FA-6005 with broad-spectrum efficacy against influenza viruses and our study presented a comprehensive study of mode of action of FA-6005 with the crystal structure of the compound in complex with NP. The influenza inhibitor holds promise as an urgently sought-after therapeutic option offering a complementary mechanism of action to existing antiviral drugs for the treatment of influenza virus infection, and that should further aid development of universal therapeutics.

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

Journal of Virology ◽

10.1128/jvi.75.17.8127-8136.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8127-8136 ◽

Cited By ~ 78

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.

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