#Influenza A and B viruses with reduced #baloxavir susceptibility display attenuated in vitro #fitness but retain ferret #transmissibility (Proc Natl Acad Sci USA, abstract)

[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]

Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility

Jeremy C. Jones, Philippe Noriel Q. Pascua, Thomas P. Fabrizio, Bindumadhav M. Marathe, Patrick Seiler, Subrata Barman, Richard J. Webby, Robert G. Webster, and Elena A. Govorkova

PNAS first published March 26, 2020 | DOI: https://doi.org/10.1073/pnas.1916825117

Contributed by Robert G. Webster, February 12, 2020 (sent for review October 2, 2019; reviewed by Rodney S. Daniels and Lieve Naesens)



The emergence of influenza viruses with reduced susceptibility to baloxavir marboxil (BXM) would limit the clinical utility of this novel antiviral. To assess the risk of such resistance emerging, we evaluated influenza A and B viruses carrying BXM-reduced susceptibility substitutions and compared their fitness to that of their drug-susceptible wild-type (I38-WT) counterparts. The 38T/F/M substitutions inhibited activity of the virus PA protein, and two of them (38T/F) hindered virus replication in cells. Even so, 38T/F/M viruses could transmit between ferrets, the gold-standard model for human transmission. These findings argue that there is a risk of transmission of BXM-resistant viruses from treated individuals. Whether such viruses could compete with WT viruses in spreading through the wider untreated community is less clear.



Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

influenza – endonuclease inhibitor – baloxavir marboxil – PA protein – I38T substitution



1 To whom correspondence may be addressed. Email: Jeremy.jones@stjude.org or robert.webster@stjude.org.

Author contributions: J.C.J., P.N.Q.P., T.P.F., R.J.W., R.G.W., and E.A.G. designed research; J.C.J., P.N.Q.P., B.M.M., and P.S. performed research; J.C.J., P.N.Q.P., T.P.F., and S.B. contributed new reagents/analytic tools; J.C.J., P.N.Q.P., T.P.F., and E.A.G. analyzed data; and J.C.J., P.N.Q.P., R.J.W., R.G.W., and E.A.G. wrote the paper.

Reviewers: R.S.D., The Francis Crick Institute; and L.N., Katholieke Universiteit Leuven.

Competing interest statement: E.A.G. reports receiving consultant fees and travel support from Genentech/Roche for serving on an advisory board. The other authors declare no conflicts of interest.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1916825117/-/DCSupplemental.

Published under the PNAS license.

Keywords: Seasonal Influenza; Antivirals; Drugs Resistance; Baloxavir.


Detection of #variants with reduced #baloxavir marboxil #susceptibility after #treatment of #children with #influenza A during the 2018/2019 influenza season (J Infect Dis., abstract)

[Source: Journal of Infectious Diseases, full page: (LINK). Abstract, edited.]

Detection of variants with reduced baloxavir marboxil susceptibility after treatment of children with influenza A during the 2018/2019 influenza season

Masatoki Sato, Emi Takashita, Masahiko Katayose, Kenji Nemoto, Nobuko Sakai, Koichi Hashimoto, Mitsuaki Hosoya

The Journal of Infectious Diseases, jiaa061, https://doi.org/10.1093/infdis/jiaa061

Published: 08 February 2020



During the 2018–2019 influenza seasons, we detected reduced baloxavir marboxil (baloxavir) susceptible variants with I38S or I38T amino acid substitutions on the PA subunit of influenza virus RNA polymerase in 7 of 18 baloxavir treated children and found that virus titer rebounded in some of these children with variants. We also found fever durations to be similar between patients with or without the variants, but the patients with variants shed the virus 3 days longer and took longer to improve clinical symptoms than those without variants.

The emergence of these variants should be monitored during future influenza seasons.

influenza, baloxavir, oseltamivir, antiviral therapy, resistance

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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Influenza A; Antivirals; Drugs Resistance; Baloxavir; Pediatrics.


#Global #Update on the Susceptibilities of #Human #Influenza Viruses to #NAIs and the #Baloxavir, 2017-2018 (Antiviral Res., abstract)

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

Antiviral Res, 104718 2020 Jan 28 [Online ahead of print]

Global Update on the Susceptibilities of Human Influenza Viruses to Neuraminidase Inhibitors and the Cap-Dependent Endonuclease Inhibitor Baloxavir, 2017-2018

Emi Takashita 1, Rod S Daniels 2, Seiichiro Fujisaki 3, Vicki Gregory 2, Larisa V Gubareva 4, Weiijuan Huang 5, Aeron C Hurt 6, Angie Lackenby 7, Ha T Nguyen 4, Dmitriy Pereyaslov 8, Merryn Roe 6, Magdi Samaan 9, Kanta Subbarao 6, Herman Tse 10, Dayan Wang 5, Hui-Ling Yen 11, Wenqing Zhang 9, Adam Meijer 12

Affiliations: 1 WHO Collaborating Centre for Reference and Research on Influenza, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo, 208-0011, Japan. Electronic address: emitaka@nih.go.jp. 2 WHO Collaborating Centre for Reference and Research on Influenza, The Francis Crick Institute, Worldwide Influenza Centre, 1 Midland Road, London, NW1 1AT, United Kingdom. 3 WHO Collaborating Centre for Reference and Research on Influenza, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo, 208-0011, Japan. 4 WHO Collaborating Centre for Surveillance, Epidemiology and Control of Influenza, Centers for Diseases Control and Prevention, 1600 Clifton RD NE, MS-G16, Atlanta, GA, 30329, USA. 5 WHO Collaborating Centre for Reference and Research on Influenza, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China. 6 WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, 3000, Australia. 7 National Infection Service, Public Health England, London, NW9 5HT, United Kingdom. 8 Division of Communicable Diseases, Health Security, & Environment, World Health Organization Regional Office for Europe, UN City, Marmorvej 51, DK-2100, Copenhagen Ø, Denmark. 9 Global Influenza Programme, World Health Organization, Avenue Appia 20, 1211, Geneva 27, Switzerland. 10 Public Health Laboratory Centre, 382 Nam Cheong Street, Hong Kong SAR, China. 11 School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China. 12 National Institute for Public Health and the Environment, PO Box 1, 3720, BA Bilthoven, the Netherlands.

PMID: 32004620 DOI: 10.1016/j.antiviral.2020.104718



The global analysis of neuraminidase inhibitor (NAI) susceptibility of influenza viruses has been conducted since the 2012-13 period. In 2018 a novel cap-dependent endonuclease inhibitor, baloxavir, that targets polymerase acidic subunit (PA) was approved for the treatment of influenza virus infection in Japan and the United States. For this annual report, the susceptibilities of influenza viruses to NAIs and baloxavir were analyzed. A total of 15409 viruses, collected by World Health Organization (WHO) recognized National Influenza Centers and other laboratories between May 2017 and May 2018, were assessed for phenotypic NAI susceptibility by five WHO Collaborating Centers (CCs). The 50% inhibitory concentration (IC50) was determined for oseltamivir, zanamivir, peramivir and laninamivir. Reduced inhibition (RI) or highly reduced inhibition (HRI) by one or more NAIs was exhibited by 0.8% of viruses tested (n = 122). The frequency of viruses with RI or HRI has remained low since this global analysis began (2012-13: 0.6%; 2013-14: 1.9%; 2014-15: 0.5%; 2015-16: 0.8%; 2016-17: 0.2%). PA gene sequence data, available from public databases (n = 13523), were screened for amino acid substitutions associated with reduced susceptibility to baloxavir (PA E23 G/K/R, PA A36V, PA A37T, PA I38 F/M/T/L, PA E119D, PA E199G): 11 (0.08%) viruses possessed such substitutions. Five of them were included in phenotypic baloxavir susceptibility analysis by two WHO CCs and IC50 values were determined. The PA variant viruses showed 6-17-fold reduced susceptibility to baloxavir. Overall, in the 2017-18 period the frequency of circulating influenza viruses with reduced susceptibility to NAIs or baloxavir was low, but continued monitoring is important.

Keywords: Baloxavir; Influenza; Neuraminidase inhibitor; Resistance; Surveillance; Susceptibility.

Copyright © 2020. Published by Elsevier B.V.

Conflict of interest statement: Declaration of competing interest None.

Keywords: Antivirals; Drugs Resistance; Influenza A; Oseltamivir; Zanamivir; Peramivir; Laninamivir; Baloxavir.


Assessing the #susceptibility of highly pathogenic #avian #influenza #H5N1 viruses to #oseltamivir using embryonated chicken #eggs (Indian J Med Res., abstract)

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

Indian J Med Res. 2019 Nov;150(5):486-491. doi: 10.4103/ijmr.IJMR_845_18.

Assessing the susceptibility of highly pathogenic avian influenza H5N1 viruses to oseltamivir using embryonated chicken eggs.

Tare DS1, Kode SS1, Hurt AC2, Pawar SD3.

Author information: 1 Avian Influenza Group, ICMR-National Institute of Virology-Microbial Containment Complex, Pune, Maharashtra, India. 2 WHO Collaborating Centre for Reference & Research on Influenza (VIDRL), Peter Doherty Institute for Infection & Immunity, Melbourne VIC 3000, Australia. 3 ICMR-National Institute of Virology-Mumbai Unit, Mumbai, Maharashtra, India.




The susceptibility of influenza viruses to neuraminidase inhibitors (NAIs) is studied using enzyme-based assays, sequence analysis and in vitro and in vivo studies. Oseltamivir carboxylate (OC) is the active prodrug of the NAI oseltamivir. There is lack of information on the use of embryonated chicken eggs for studying susceptibility of highly pathogenic avian influenza (HPAI) H5N1 viruses to antiviral drugs. The aim of the present study was to assess the use of 10 day old embryonated chicken eggs for studying antiviral susceptibility of HPAI H5N1 viruses.


Two HPAI H5N1 viruses isolated from India were used in the study. Fluorescence-based NAI assay was performed to determine antiviral susceptibility of these viruses. In ovo antiviral assays were carried out using 10 day old embryonated chicken eggs. The virus dilutions were incubated with 14 μg/ml of OC and inoculated in the allantoic cavity. In the eggs, 50 per cent egg infectious dose (EID50) titres as well as mortality were quantitated.


The two viruses used were susceptible to OC in the NAI assay. It was found that there was a significant drop in EID50titres; however, no significant protection from mortality after OC treatment was observed.


By measuring viral titres, the egg model was suitable to study the susceptibility of HPAI viruses to antiviral drugs along with NAI assay. The present study highlights the use of eggs as a model to study susceptibility of HPAI viruses to OC.

KEYWORDS: Antivirals – embryonated chicken eggs – H5N1 – highly pathogenic avian influenza – oseltamivir

PMID: 31939392 DOI: 10.4103/ijmr.IJMR_845_18

Keywords: Antivirals; Drugs resistance; Avian Influenza; H5N1; Animal models.


In vitro Evaluation of #Antifungal Drug #Combinations against #MDR #Candida auris isolates from #NY #Outbreak (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

In vitro Evaluation of Antifungal Drug Combinations against Multidrug-resistant Candida auris isolates from New York Outbreak

Brittany O’Brien, Sudha Chaturvedi, Vishnu Chaturvedi

DOI: 10.1128/AAC.02195-19



Since 2016, New York hospitals and healthcare facilities have faced an unprecedented outbreak of the pathogenic yeast Candida auris. We tested over one thousand C. auris isolates from affected facilities and found high-resistance to fluconazole (MIC > 256 mg/L), and variable resistance to other antifungal drugs. Therefore, we tested if two-drug combinations are effective in vitro against multidrug-resistant C. auris. Broth micro-dilution antifungal combination plates were custom-manufactured by TREK Diagnostic System. We used 100% inhibition endpoints for the drug combination as reported earlier for the intra- and inter-laboratory agreements against Candida species. The results were derived from 12,960 readings, for fifteen C. auris isolates tested against 864 two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/L potentiated the most combinations. For nine C. auris isolates resistant to amphotericin B (AMB, MIC ≥ 2.0 mg/L]), AMB/5FC (0.25/1.0 mg/L) yielded 100% inhibition. Six C. auris isolates resistant to three echinocandins (anidulafungin [AFG, MIC ≥ 4.0 mg/L], caspofungin [CAS, MIC ≥ 2.0 mg/L], and micafungin [MFG, MIC ≥ 4.0 mg/L]), were 100% inhibited by AFG/5FC and CAS/5FC (0.0078/1 mg/L), and MFG/5FC (0.12/1 mg/L). None of the combinations were effective for C. auris 18-1 and 18-13 (FLC > 256 mg/L, 5FC > 32 mg/L) except MFG/5FC (0.1/0.006 mg/L). Thirteen isolates with high voriconazole MIC (VRC, > 2 mg/L) were 100% inhibited by the VRC/5FC (0.015/1 mg/L). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to manage multidrug-resistant C. auris.

Copyright © 2020 American Society for Microbiology. All Rights Reserved.

Keywords: Drugs Resistance; Candida auris; USA.


#Evolution of highly pathogenic #H7N3 #avian #influenza viruses in #Mexico (Zoonoses Public Health, abstract)

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

Zoonoses Public Health. 2020 Jan 7. doi: 10.1111/zph.12673. [Epub ahead of print]

Evolution of highly pathogenic H7N3 avian influenza viruses in Mexico.

Trovão NS1, Talavera GA2, Nelson MI1, Perez de la Rosa JD3.

Author information: 1 Fogarty International Center, National Institutes of Health, Bethesda, Maryland. 2 Universidad Autónoma de Yucatán, Yucatán, Mexico. 3 Centro Nacional de Servicios de Constatación en Salud Animal (CENAPA), Morelos, Mexico.



Highly pathogenic H7N3 influenza A viruses have persisted in poultry in Mexico since 2012, diversifying into multiple lineages that have spread to three Mexican states, as of 2016. The H7N3 viruses segregate into three distinct clades that are geographically structured. All 2016 viruses are resistant to adamantane antiviral drugs and have an extended 24-nucleotide insertion at the HA cleavage site that was acquired from host 28S ribosomal RNA.

Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

KEYWORDS: BEAST; H7N3; H7N3 subtype; avian influenza virus; evolutionary dynamics; influenza in birds; phylogenetics

PMID: 31912652 DOI: 10.1111/zph.12673

Keywords: Avian Influenza; H7N3; Poultry; Mexico; Antivirals; Drugs Resistance; Amantadine.


#NDM-β-Lactamase-5–Producing #Escherichia coli in Companion #Animals, #USA (Emerg Infect Dis., abstract)

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

Volume 26, Number 2—February 2020 / Research Letter

New Delhi Metallo-β-Lactamase-5–Producing Escherichia coli in Companion Animals, United States

Stephen D. Cole, Laura Peak, Gregory H. Tyson, Renate Reimschuessel, Olgica Ceric, and Shelley C. Rankin

Author affiliations: University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA (S.D. Cole, S.C. Rankin); Louisiana State University, Baton Rouge, Louisiana USA (L. Peak); US Food and Drug Administration, Silver Spring, Maryland, USA (G.H. Tyson, R. Reimscheussel, O. Ceric)



We report isolation of a New Delhi metallo-β-lactamase-5–producing carbapenem-resistant Escherichia coli sequence type 167 from companion animals in the United States. Reports of carbapenem-resistant Enterobacteriaceae in companion animals are rare. We describe a unique cluster of blaNDM-5–producing E. coli in a veterinary hospital.

Keywords: Antibiotics; Drugs Resistance; Carbapenem; Beta-lactams; NDM1; USA.