#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.


The effect of #mutations derived from mouse-adapted #H3N2 seasonal #influenza A virus to #pathogenicity and #host adaptation (PLOS One, abstract)

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


The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation

Eun-Ji Choi , Young Jae Lee , Jin-Moo Lee, Yeon-Jung Kim, Jang-Hoon Choi, Byeongwoo Ahn, Kisoon Kim, Myung Guk Han


Published: January 9, 2020 / DOI: https://doi.org/10.1371/journal.pone.0227516



Elucidating the genetic basis of influenza A viruses (IAVs) is important to understand which mutations will determine the virulence and the host range of mammals. Here, seasonal H3N2 influenza was adapted in mice by serial passage and four mutants, each carrying amino acid substitutions related to mouse adaptation in either the PB2, HA, NP, or NA protein, were generated. To confirm the contribution of each gene to enhanced pathogenicity and mouse adaptation, mice were inoculated with the respective variants, and virulence, replication, histopathology, and infectivity were examined. The virus harboring HA mutations displayed increased infection efficiency and replication competence, resulting in higher mortality in mice relative to those infected with wild-type virus. By contrast, the NP D34N mutation caused rapid and widespread infection in multiple organs without presenting virulent symptoms. Additionally, the PB2 F323L mutation presented delayed but elevated replication competence in the respiratory tract, whereas the S331R mutation in NA showed no considerable effects on mouse adaptation. These results suggested that mouse-adapted changes in HA are major factors in increased pathogenicity and that mutations in NP and PB2 also contribute to cross-species adaptability. Our findings offer a better understanding of the molecular basis for IAV pathogenicity and adaptation in a new host.


Citation: Choi E-J, Lee YJ, Lee J-M, Kim Y-J, Choi J-H, Ahn B, et al. (2020) The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation. PLoS ONE 15(1): e0227516. https://doi.org/10.1371/journal.pone.0227516

Editor: Man-Seong Park, Korea University College of Medicine and School of Medicine, REPUBLIC OF KOREA

Received: August 7, 2019; Accepted: December 19, 2019; Published: January 9, 2020

Copyright: © 2020 Choi 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: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by grants of the National Institute of Health, Korea (2017-NI43001 and 2014-ER4301-02). The funder provided experiment resources, equipment, reagents and space, and helped to complete manuscript by pay the cost for English proofreading service, http://www.cdc.go.kr/index.es?sid=a5. The funder 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: Influenza A; H3N2; Viral pathogenesis; Animal models.


#Genetic #variations on 31 and 450 residues of #influenza A #nucleoprotein affect viral #replication and translation (J Biomed Sci., abstract)

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

J Biomed Sci. 2020 Jan 6;27(1):17. doi: 10.1186/s12929-019-0612-z.

Genetic variations on 31 and 450 residues of influenza A nucleoprotein affect viral replication and translation.

Hung SJ1, Hsu YM1, Huang SW2, Tsai HP1,3, Lee LYY4, Hurt AC4, Barr IG4, Shih SR5, Wang JR6,7,8,9.

Author information: 1 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 701, Taiwan. 2 National Mosquito-Borne Diseases Control Research Center, National Health Research Institutes, Tainan, Taiwan. 3 Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan. 4 WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, 3000, Australia. 5 Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 6 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 701, Taiwan. jrwang@mail.ncku.edu.tw. 7 Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan. jrwang@mail.ncku.edu.tw. 8 Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan. jrwang@mail.ncku.edu.tw. 9 National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan. jrwang@mail.ncku.edu.tw.




Influenza A viruses cause epidemics/severe pandemics that pose a great global health threat. Among eight viral RNA segments, the multiple functions of nucleoprotein (NP) play important roles in viral replication and transcription.


To understand how NP contributes to the virus evolution, we analyzed the NP gene of H3N2 viruses in Taiwan and 14,220 NP sequences collected from Influenza Research Database. The identified genetic variations were further analyzed by mini-genome assay, virus growth assay, viral RNA and protein expression as well as ferret model to analyze their impacts on viral replication properties.


The NP genetic analysis by Taiwan and global sequences showed similar evolution pattern that the NP backbones changed through time accompanied with specific residue substitutions from 1999 to 2018. Other than the conserved residues, fifteen sporadic substitutions were observed in which the 31R, 377G and 450S showed higher frequency. We found 31R and 450S decreased polymerase activity while the dominant residues (31 K and 450G) had higher activity. The 31 K and 450G showed better viral translation and replication in vitro and in vivo.


These findings indicated variations identified in evolution have roles in modulating viral replication in vitro and in vivo. This study demonstrates that the interaction between variations of NP during virus evolution deserves future attention.

KEYWORDS: Evolution; Ferret study; H3N2; Influenza virus; Nucleoprotein; Viral replication; Viral translation

PMID: 31906961 DOI: 10.1186/s12929-019-0612-z

Keywords: Influenza A; H3N2; Viral pathogenesis.


#H3N2 #avian #influenza viruses detected in live #poultry #markets in #China bind to #human-type #receptors and transmit in guinea pigs and ferrets (Emerg Microbes Infect., abstract)

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

Emerg Microbes Infect. 2019;8(1):1280-1290. doi: 10.1080/22221751.2019.1660590.

H3N2 avian influenza viruses detected in live poultry markets in China bind to human-type receptors and transmit in guinea pigs and ferrets.

Guan L1, Shi J1, Kong X1, Ma S1, Zhang Y1, Yin X1, He X1, Liu L1, Suzuki Y2, Li C1, Deng G1, Chen H1.

Author information: 1 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS , Harbin , People’s Republic of China. 2 College of Life and Health Sciences, Chubu University , Aichi , Japan.



The H3N2 influenza viruses became widespread in humans during the 1968 H3N2 pandemic and have been a major cause of influenza epidemics ever since. Different lineages of H3N2 influenza viruses are also commonly found in animals. If a different lineage of H3N2 virus jumps to humans, a human influenza pandemic could occur with devastating consequences. Here, we studied the genetics, receptor-binding properties, and replication and transmission in mammals of 15 H3N2 avian influenza viruses detected in live poultry markets in China. We found that the H3N2 avian influenza viruses are complicated reassortants with distinct replication phenotypes in mice. Five viruses replicated efficiently in mice and bound to both human-type and avian-type receptors. These viruses transmitted efficiently to direct-contact guinea pigs, and three of them also transmitted among guinea pigs and ferrets via respiratory droplets. Moreover, ferret antiserum induced by human H3N2 viruses did not react with any of the H3N2 avian influenza viruses. Our study demonstrates that the H3N2 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H3N2 influenza viruses circulating in nature.

KEYWORDS: Avian influenza virus; H3N2; ferret; guinea pig; transmission

PMID: 31495283 PMCID: PMC6746299 DOI: 10.1080/22221751.2019.1660590 [Indexed for MEDLINE] Free PMC Article

Keywords: Avian Influenza; H3N2; Reassortant strain; Poultry; China; Animal models.


#Genetic and #serologic #surveillance of #canine (CIV) and #equine (EIV) #influenza virus in Nuevo León State, #México (PeerJ., abstract)

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

PeerJ. 2019 Dec 17;7:e8239. doi: 10.7717/peerj.8239. eCollection 2019.

Genetic and serologic surveillance of canine (CIV) and equine (EIV) influenza virus in Nuevo León State, México.

Plata-Hipólito CB1, Cedillo-Rosales S2, Obregón-Macías N3, Hernández-Luna CE4, Rodríguez-Padilla C1, Tamez-Guerra RS1, Contreras-Cordero JF1.

Author information: 1 Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Laboratorio de Inmunología y Virología, San Nicolás de los Garza, Nuevo León, México. 2 Universidad Autónoma de Nuevo León, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Virología, Escobedo, Nuevo León, México. 3 Universidad Autónoma de Nuevo León, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Grandes Especies, Escobedo, Nuevo León, México. 4 Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Química, San Nicolás de los Garza, Nuevo León, México.




Despite the uncontrolled distribution of the Influenza A virus through wild birds, the detection of canine influenza virus and equine influenza virus in Mexico was absent until now. Recently, outbreaks of equine and canine influenza have been reported around the world; the virus spreads quickly among animals and there is potential for zoonotic transmission.


Amplification of the Influenza A virus matrix gene from necropsies, nasal and conjunctival swabs from trash service horses and pets/stray dogs was performed through RT-PCR. The seroprevalence was carried out through Sandwich enzyme-linked immunosorbent assay system using the M1 recombinant protein and polyclonal antibodies anti-M1.


The matrix gene was amplified from 13 (19.11%) nasal swabs, two (2.94%) conjunctival swabs and five (7.35%) lung necropsies, giving a total of 20 (29.41%) positive samples in a pet dog population. A total of six (75%) positive samples of equine nasal swab were amplified. Sequence analysis showed 96-99% identity with sequences of Influenza A virus matrix gene present in H1N1, H1N2 and H3N2 subtypes. The phylogenetic analysis of the sequences revealed higher identity with matrix gene sequences detected from zoonotic isolates of subtype H1N1/2009. The detection of anti-M1 antibodies in stray dogs showed a prevalence of 123 (100%) of the sampled population, whereas in horses, 114 (92.68%) positivity was obtained.


The results unveil the prevalence of Influenza A virus in the population of horses and dogs in the state of Nuevo Leon, which could indicate a possible outbreak of equine and Canine Influenza in Mexico. We suggest that the prevalence of Influenza virus in companion animals be monitored to investigate its epizootic and zoonotic potential, in addition to encouraging the regulation of vaccination in these animal species in order to improve their quality of life.

© 2019 Plata-Hipólito et al.

KEYWORDS: Canine Influenza Virus (CIV); Equine Influenza Virus (EIV); Matrix gene (M); Polyclonal antibodies

PMID: 31871842 PMCID: PMC6924343 DOI: 10.7717/peerj.8239

Keywords: Influenza A; Equine Influenza; Canine Avian Influenza; H1N1pdm09; H1N2; H3N2; H1N1; Reassortant strains; Dogs; Horses; Mexico; Serology.


#Surveillance of #swine #influenza viruses in sentinel #familial #farms in Hung Yen province in Northern #Vietnam in 2013-2014 (Zoonoses Public Health, abstract)

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

Zoonoses Public Health. 2019 Dec 19. doi: 10.1111/zph.12671. [Epub ahead of print]

Surveillance of swine influenza viruses in sentinel familial farms in Hung Yen province in Northern Vietnam in 2013-2014.

Baudon E1,2, Peyre M2, Tung DD3, Thi Nga P3, Khong NV3, Cowling BJ1, Peiris M1.

Author information: 1 The University of Hong Kong, Hong Kong, China. 2 French Agricultural Research Center for International Development (CIRAD), Montpellier, France. 3 National Institute of Veterinary Research, Hanoi, Vietnam.



From May 2013 to April 2014, 15 swine family-run farms (17 pig litters) in two districts in Hung Yen province, near Hanoi, were virologically and epizootiologically monitored for swine influenza viruses (SIV) monthly. No SIV was isolated from nasal swabs. Maternal antibodies were detected in 10 litters, and seroconversion against SIV was detected in six litters. There was a marked difference in patterns of SIV transmission in the two districts. Van Lam district which has low density of swine with mainly smallholder farms had low intensity of SIV, with much of the infection caused by H1N1 2009 pandemic-like viruses A(H1N1)pdm09, likely originated from humans. In contrast, Van Giang district, which has high swine density and larger farms, had high levels of typical SIV (triple reassortants H3N2 and H3N2 Binh Duong lineage viruses) circulating within swine. With one exception, the SIV lineages detected were those we concurrently isolated from studies in a large central abattoir in Hanoi. Influenza-like illness symptoms reported by farmers were poorly correlated with serological evidence of SIV infection.

© 2019 Blackwell Verlag GmbH.

KEYWORDS: Vietnam; familial farm; maternal antibody; surveillance; swine influenza; value chain

PMID: 31855326 DOI: 10.1111/zph.12671

Keyword: Swine Influenza; Influenza A; H1N1pdm09; H3N2; Serology; Pigs; Vietnam.


#Childhood immune #imprinting to #influenza A shapes birth year-specific #risk during seasonal #H1N1 and #H3N2 #epidemics (PLOS Pathog., abstract)

[Source: PLOS Pathogens, full page: (LINK). Abstract, edited.]


Childhood immune imprinting to influenza A shapes birth year-specific risk during seasonal H1N1 and H3N2 epidemics

Katelyn M. Gostic , Rebecca Bridge, Shane Brady, Cécile Viboud, Michael Worobey, James O. Lloyd-Smith


Published: December 19, 2019 / DOI: https://doi.org/10.1371/journal.ppat.1008109



Across decades of co-circulation in humans, influenza A subtypes H1N1 and H3N2 have caused seasonal epidemics characterized by different age distributions of cases and mortality. H3N2 causes the majority of severe, clinically attended cases in high-risk elderly cohorts, and the majority of overall deaths, whereas H1N1 causes fewer deaths overall, and cases shifted towards young and middle-aged adults. These contrasting age profiles may result from differences in childhood imprinting to H1N1 and H3N2 or from differences in evolutionary rate between subtypes. Here we analyze a large epidemiological surveillance dataset to test whether childhood immune imprinting shapes seasonal influenza epidemiology, and if so, whether it acts primarily via homosubtypic immune memory or via broader, heterosubtypic memory. We also test the impact of evolutionary differences between influenza subtypes on age distributions of cases. Likelihood-based model comparison shows that narrow, within-subtype imprinting shapes seasonal influenza risk alongside age-specific risk factors. The data do not support a strong effect of evolutionary rate, or of broadly protective imprinting that acts across subtypes. Our findings emphasize that childhood exposures can imprint a lifelong immunological bias toward particular influenza subtypes, and that these cohort-specific biases shape epidemic age distributions. As a consequence, newer and less “senior” antibody responses acquired later in life do not provide the same strength of protection as responses imprinted in childhood. Finally, we project that the relatively low mortality burden of H1N1 may increase in the coming decades, as cohorts that lack H1N1-specific imprinting eventually reach old age.


Author summary

Influenza viruses of subtype H1N1 and H3N2 both cause seasonal epidemics in humans, but with different age-specific impacts. H3N2 causes a greater proportion of cases in older adults than H1N1, and more deaths overall. People tend to gain the strongest immune memory of influenza viruses encountered in childhood, and so differences in H1N1 and H3N2’s age-specific impacts may reflect that individuals born in different eras of influenza circulation have been imprinted with different immunological risk profiles. Another idea is that H3N2 may be more able to infect immunologically experienced adults because it evolves slightly faster than H1N1 and can more quickly escape immune memory. We analyzed a large epidemiological data set and found that birth year-specific differences in childhood immune imprinting, not differences in evolutionary rate, explain differences in H1N1 and H3N2’s age-specific impacts. These results can help epidemiologists understand how epidemic risk from specific influenza subtypes is distributed across the population and predict how population risk may shift as differently imprinted birth years grow older. Further, these results provide immunological clues to which facets of immune memory become biased in childhood, and then later play a strong role in protection during seasonal influenza epidemics.


Citation: Gostic KM, Bridge R, Brady S, Viboud C, Worobey M, Lloyd-Smith JO (2019) Childhood immune imprinting to influenza A shapes birth year-specific risk during seasonal H1N1 and H3N2 epidemics. PLoS Pathog 15(12): e1008109. https://doi.org/10.1371/journal.ppat.1008109

Editor: Sabra L. Klein, Johns Hopkins Bloomberg School of Public Health, UNITED STATES

Received: July 9, 2019; Accepted: September 25, 2019; Published: December 19, 2019

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are available as Supporting Information files. All data are also archived alongside the full suite of code used to perform analyses and generate plots, at https://zenodo.org/badge/latestdoi/160883450.

Funding: KMG was supported by the National Institutes of Health (F31AI134017, T32-GM008185). JOLS was supported by NSF grants OCE-1335657 and DEB-1557022, SERDP RC-2635, and DARPA PREEMPT D18AC00031. MW was supported by the David and Lucile Packard Foundation. 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: Seasonal Influenza; Immunology; H1N1; H3N2.