#Growth activation of #influenza virus by #trypsin and effect of T-705 (#favipiravir) on trypsin-optimized growth condition (Acta Virol., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Acta Virol. 2019;63(3):309-315. doi: 10.4149/av_2019_311.

Growth activation of influenza virus by trypsin and effect of T-705 (favipiravir) on trypsin-optimized growth condition.

Daikoku T, Okuda T, Kawai M, Morita N, Tanaka T, Takemoto M, Fukuda Y, Takahashi K, Nomura N, Shiraki K.



Influenza virus is activated by proteolytic cleavage of hemagglutinin by trypsin. After determining the optimal trypsin concentration, intracellular and extracellular influenza A/PR/8/34 (H1N1) and A/Victoria/361/2011 (H3N2) virus productions were compared in cultures treated with T-705 (favipiravir) and GS 4071 (an active form of oseltamivir). Although both drugs efficiently inhibited extracellular viral RNA release in a dose-dependent manner, T-705 inhibited it to the level of the inoculum without trypsin treatment, while GS 4071 inhibited it to a final level 10 times higher than that without trypsin. T-705 inhibited intracellular viral RNA production to the level of input virus in both trypsin-treated and untreated cells. In contrast, GS 4071 dose-dependently inhibited intracellular viral RNA production in cells treated with trypsin but allowed viral RNA synthesis. The level of maximum inhibition by GS 4071 was 10 times higher than that of cells without trypsin and 1,000 times greater than the inoculum titer in cells without trypsin. T-705 inhibited both intracellular and extracellular virus production 1,000 and 10 times more strongly, respectively, than GS 4071. T-705 has powerful anti-influenza activity in the absence of trypsin and even in the trypsin-optimized growth condition, suggesting the therapeutic advantage in treatment of influenza complicated with bacterial pneumonia.

Keywords: influenza; T-705; Tamiflu; trypsin; bacterial trypsin-like protease.

PMID: 31507197 DOI: 10.4149/av_2019_311

Keywords: Influenza A; H1N1; H3N2; Antivirals; Favipiravir; Oseltamivir.



Susceptibility of #Influenza A, B, C, and D Viruses to #Baloxavir (Emerg Infect Dis., abstract)

[Source: US Centers for Disease Control and Prevention (CDC), Emerging Infectious Diseases Journal, full page: (LINK). Abstract, edited.]

Volume 25, Number 10—October 2019 / Dispatch

Susceptibility of Influenza A, B, C, and D Viruses to Baloxavir

Vasiliy P. Mishin, Mira C. Patel, Anton Chesnokov, Juan De La Cruz, Ha T. Nguyen, Lori Lollis, Erin Hodges, Yunho Jang, John Barnes, Timothy Uyeki, Charles T. Davis, David E. Wentworth, and Larisa V. Gubareva

Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (V.P. Mishin, M.C. Patel, A. Chesnokov, J. De La Cruz, H.T. Nguyen, L. Lollis, E. Hodges, Y. Jang, J. Barnes, T. Uyeki, C.T. Davis, D.E. Wentworth, L.V. Gubareva); Battelle Memorial Institute, Atlanta (M.C. Patel, J. De La Cruz, H.T. Nguyen, L. Lollis)



Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2–98.3 nmol/L); susceptibility pattern was influenza A ˃ B ˃ C ˃ D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.

Keywords: Antivirals; Drugs Resistance; Oseltamivir; Favipiravir; Baloxavir; Influenza A; Influenza B; Influenza C; Influenza D; H1N1pdm09; H3N2; H7N9.


#Combination #Therapy With #Neuraminidase and #Polymerase #Inhibitors in Nude Mice Infected With #Influenza Virus (J Infect Dis., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

J Infect Dis. 2018 Mar 5;217(6):887-896. doi: 10.1093/infdis/jix606.

Combination Therapy With Neuraminidase and Polymerase Inhibitors in Nude Mice Infected With Influenza Virus.

Kiso M1, Lopes TJS1,2, Yamayoshi S1, Ito M1, Yamashita M1, Nakajima N3, Hasegawa H3, Neumann G2, Kawaoka Y1,2,4.

Author information: 1 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Japan. 2 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison. 3 Department of Pathology, National Institute of Infectious Diseases, Tokyo. 4 ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan.




Treatment of immunocompromised, influenza virus-infected patients with the viral neuraminidase inhibitor oseltamivir often leads to the emergence of drug-resistant variants. Combination therapy with compounds that target different steps in the viral life cycle may improve treatment outcomes and reduce the emergence of drug-resistant variants.


Here, we infected immunocompromised nude mice with an influenza A virus and treated them with neuraminidase (oseltamivir, laninamivir) or viral polymerase (favipiravir) inhibitors, or combinations thereof.


Combination therapy for 28 days increased survival times compared with monotherapy, but the animals died after treatment was terminated. Mono- and combination therapies did not consistently reduce lung virus titers. Prolonged viral replication led to the emergence of neuraminidase inhibitor-resistant variants, although viruses remained sensitive to favipiravir. Overall, favipiravir provided greater benefit than neuraminidase inhibitors.


Collectively, our data demonstrate that combination therapy in immunocompromised hosts increases survival times, but does not suppress the emergence of neuraminidase inhibitor-resistant variants.

PMID: 29186472 DOI: 10.1093/infdis/jix606 [Indexed for MEDLINE]

Keywords: Antivirals; Drugs Resistance; Oseltamivir; Laninamivir; Favipiravir; Influenza A; Animal models.


#Prodrugs of the Phosphoribosylated Forms of Hydroxypyrazinecarboxamide Pseudobase #T705 and Its De-Fluoro Analogue T-1105 as Potent #Influenza Virus #Inhibitors (J Med Chem., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

J Med Chem. 2018 Jul 26;61(14):6193-6210. doi: 10.1021/acs.jmedchem.8b00617. Epub 2018 Jul 3.

Prodrugs of the Phosphoribosylated Forms of Hydroxypyrazinecarboxamide Pseudobase T-705 and Its De-Fluoro Analogue T-1105 as Potent Influenza Virus Inhibitors.

Huchting J1,2, Vanderlinden E2, Winkler M1, Nasser H1, Naesens L2, Meier C1.

Author information: 1 Organic Chemistry, Department of Chemistry, Faculty of Sciences, Hamburg University , Martin-Luther-King-Platz 6 , D-20146 Hamburg , Germany. 2 KU Leuven, Rega Institute for Medical Research , Herestraat 49 , B-3000 Leuven , Belgium.



We here disclose chemical synthesis of ribonucleoside 5′-monophosphate (RMP), -diphosphate (RDP), and -triphosphate (RTP) and cycloSal-, Di PPro-, and Tri PPPro nucleotide prodrugs of the antiviral pseudobase T-1105. Moreover, we include one nucleoside diphosphate prodrug of the chemically less stable T-705. We demonstrate efficient T-1105-RDP and -RTP release from the Di PPro and Tri PPPro compounds by esterase activation. Using crude enzyme extracts, we saw rapid phosphorylation of T-1105-RDP into T-1105-RTP. In sharp contrast, phosphorylation of T-1105-RMP was not seen, indicating a yet unrecognized bottleneck in T-1105’s metabolic activation. Accordingly, Di PPro and Tri PPPro compounds displayed improved cell culture activity against influenza A and B virus, which they retained in a mutant cell line incapable of activating the nucleobase parent. T-1105-RTP had a strong inhibitory effect against isolated influenza polymerase, and Di PPro-T-1105-RDP showed 4-fold higher potency in suppressing one-cycle viral RNA synthesis versus T-1105. Hence, our T-1105-RDP and -RTP prodrugs improve antiviral potency and achieve efficient metabolic bypass.

PMID: 29906392 DOI: 10.1021/acs.jmedchem.8b00617 [Indexed for MEDLINE]

Keywords: Antivirals; Favipiravir; Influenza A.


Establishment of a #Cell #Culture Model of Persistent #Flaviviral #Infection: #Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides (Viruses, abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Viruses. 2019 Jun 17;11(6). pii: E560. doi: 10.3390/v11060560.

Establishment of a Cell Culture Model of Persistent Flaviviral Infection: Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides.

Sempere RN1,2, Arias A3.

Author information: 1 Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark. rnsempere@abiopep.com. 2 Abiopep Sociedad Limitada, Parque Científico de Murcia, 30100 Murcia, Spain. rnsempere@abiopep.com. 3 Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark. aaesteban2@gmail.com.



Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5′ end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.

KEYWORDS: antiviral therapies; chronic viral infection; defective viral genomes; emerging arboviruses; lethal mutagenesis

PMID: 31212939 DOI: 10.3390/v11060560

Keywords: Flavivirus; Usutu virus; Antivirals; Favipiravir; Ribavirin.


T-705 [#favipiravir] induces lethal #mutagenesis in #Ebola and #Marburg populations in macaques (Antiviral Res., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Antiviral Res. 2019 Jun 10. pii: S0166-3542(19)30310-9. doi: 10.1016/j.antiviral.2019.06.001. [Epub ahead of print]

T-705 induces lethal mutagenesis in Ebola and Marburg populations in macaques.

Espy N1, Nagle E1, Pfeffer B1, Garcia K1, Chitty AJ1, Wiley M1, Sanchez-Lockhart M1, Bavari S1, Warren T1, Palacios G2.

Author information: 1 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA. 2 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA. Electronic address: gustavo.f.palacios.ctr@mail.mil.



Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.

Copyright © 2019. Published by Elsevier B.V.

PMID: 31195019 DOI: 10.1016/j.antiviral.2019.06.001

Keywords: Filovirus; Antivirals; Favipiravir; Animal models.


Therapeutic efficacy of #favipiravir against #Bourbon virus in mice (PLoS One, abstract)

[Source: PLoS One, full page: (LINK). Abstract, edited.]


Therapeutic efficacy of favipiravir against Bourbon virus in mice

Traci L. Bricker, Md. Shafiuddin, Anshu P. Gounder, Andrew B. Janowski, Guoyan Zhao, Graham D. Williams, Brett W. Jagger, Michael S. Diamond, Thomas Bailey, Jennie H. Kwon, David Wang, Adrianus C. M. Boon

Published: June 13, 2019 / DOI: https://doi.org/10.1371/journal.ppat.1007790



Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV.


Author summary

Bourbon virus (BRBV) is a novel tick-borne RNA virus that can cause fatal disease in humans. No approved antiviral treatment is available. We have cultured the second human isolate of BRBV and with it developed a small animal disease model. In this mouse model, BRBV causes severe disease as measured by weight loss after infection and uniform death 6 to 10 days after infection. Virus replication occurred predominantly in the spleen and the liver of the infected animals, with additional organs infected at later time points after infection. This disease model was used to test the efficacy of favipiravir, a viral RNA polymerase inhibitor that was developed for the related Influenza A virus. Prophylactic and therapeutic treatment with favipiravir resulted in complete protection from a lethal BRBV infection. These data suggest that favipiravir and perhaps other RNA polymerase inhibitors could be used to treat BRBV infections in humans.


Citation: Bricker TL, Shafiuddin M, Gounder AP, Janowski AB, Zhao G, Williams GD, et al. (2019) Therapeutic efficacy of favipiravir against Bourbon virus in mice. PLoS Pathog 15(6): e1007790. https://doi.org/10.1371/journal.ppat.1007790

Editor: Sonja Best, National Institute of Allergy and Infectious Diseases, UNITED STATES

Received: February 6, 2019; Accepted: April 26, 2019; Published: June 13, 2019

Copyright: © 2019 Bricker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The genome sequence of Bourbon virus: All sequence files are available from the GenBank database (accession number(s) MK453524-MK453529). All other relevant data are within the manuscript and its supporting information files.

Funding: The work was in part funded by R01-AI118938, and R21-AI137450 and the Children’s Discovery Institute grant PD-II-2018-702. APG and GDW were supported by the Infectious Disease Training Grant (T32 AI007172). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Keywords: Bourbon virus; Viral pathogenesis; Antivirals; Favipiravir; Animal models.