#H7N9 #Influenza Virus Containing a #Polybasic HA Cleavage Site Requires Minimal Host #Adaptation to Obtain a Highly Pathogenic Disease Phenotype in Mice (Viruses, abstract)

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

Viruses. 2020 Jan 5;12(1). pii: E65. doi: 10.3390/v12010065.

H7N9 Influenza Virus Containing a Polybasic HA Cleavage Site Requires Minimal Host Adaptation to Obtain a Highly Pathogenic Disease Phenotype in Mice.

Chan M1, Leung A1, Hisanaga T2, Pickering B2,3, Griffin BD1,3, Vendramelli R1, Tailor N1, Wong G4,5, Bi Y6, Babiuk S2, Berhane Y2, Kobasa D1,3.

Author information: 1 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB R3E 3R2, Canada. 2 National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, Canada. 3 Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, MB R3E 0J9, Canada. 4 Institut Pasteur of Shanghai, Chinese Academy of Sciences, Life Science Research Building 320 Yueyang Road, Xuhui District, Shanghai 200031, China. 5 Département de microbiologie-infectiologie et d’immunologie, Université Laval, 1050 avenue de la Médecine, QC G1V 0A6, Canada. 6 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing 100101, China.



Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease; however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species.

KEYWORDS: H7N9; HPAI; influenza virus; mammalian adaptation; mice; polybasic HA

PMID: 31948040 DOI: 10.3390/v12010065

Keywords: Avian Influenza; H7N9; Viral pathogenesis; Animal models.


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.


Identifying target cells for a #tick-borne virus that causes #fatal #hemorrhagic fever (J Clin Invest., abstract)

[Source: Journal of Clinical Investigation, full page: (LINK). Abstract, edited.]

Identifying target cells for a tick-borne virus that causes fatal hemorrhagic fever

Satoko Yamaoka, Carla Weisend, and Hideki Ebihara

First published January 6, 2020


Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease in China, South Korea, and Japan caused by the tick-borne SFTS virus (SFTSV). Severe and fatal SFTS presents as a hemorrhagic fever characterized by high viral load, uncontrolled inflammatory response, dysregulated adaptive immunity, coagulation abnormalities, hemorrhage, and multiorgan failure with up to 33% case fatality rates (CFRs). Despite its public health significance in Asia, vaccines and specific therapeutics against SFTS are still unavailable. A better understanding of the pathogenesis of SFTS is crucial to improving medical countermeasures against this devastating disease. In this issue of the JCI, Suzuki and colleagues analyzed histopathological samples from 22 individuals who succumbed to SFTS, and identified antibody-producing B cell–lineage plasmablasts and macrophages as principal target cells for SFTSV infection in fatal SFTS. Their results suggest that SFTSV-infected post–germinal center B cells, plasmablasts, and macrophages affect systemic immunopathology and dysregulation, which likely leads to fatal outcomes.

Keywords: SFTS; Immunopathology; Viral pathogenesis.


#SFTS virus targets B cells in lethal #human #infections (J Clin Invest., abstract)

[Source: Journal of Clinical Investigation, full page: (LINK). Abstract, edited.]

Severe fever with thrombocytopenia syndrome virus targets B cells in lethal human infections

Tadaki Suzuki,1 Yuko Sato,1 Kaori Sano,1,2 Takeshi Arashiro,1 Harutaka Katano,1 Noriko Nakajima,1 Masayuki Shimojima,4 Michiyo Kataoka,1 Kenta Takahashi,1 Yuji Wada,1 Shigeru Morikawa,3 Shuetsu Fukushi,4 Tomoki Yoshikawa,4 Masayuki Saijo,4 and Hideki Hasegawa1,2,5

First published January 6, 2020

Related article: Identifying target cells for a tick-borne virus that causes fatal hemorrhagic fever, Satoko Yamaoka, … , Carla Weisend, Hideki Ebihara

Category: Commentary



Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by a tick-borne banyangvirus and is associated with high fatality. Despite increasing incidence of SFTS and serious public health concerns in East Asia, the pathogenesis of lethal SFTS virus (SFTSV) infection in humans is not fully understood. Numbers of postmortem examinations to determine target cells of the viral infection have so far been limited. Here we showed that B cells differentiating into plasmablasts and macrophages in secondary lymphoid organs were targets for SFTSV at the end stage of lethal infection, and the majority of SFTSV-infected cells were B cell–lineage lymphocytes. In affected individuals, B cell–lineage lymphocytes with SFTSV infection were widely distributed in both lymphoid and nonlymphoid organs, and infiltration of these cells into the capillaries of the organs could be observed occasionally. Moreover, a human plasmablastic lymphoma cell line, PBL-1, was susceptible to SFTSV propagation, and had a similar immunophenotype to that of target cells of SFTSV in fatal SFTS. PBL-1 can therefore provide a potential in vitro model for human SFTSV infection. These results extend our understanding of the pathogenesis of human lethal SFTSV infection, and can facilitate the development of SFTSV countermeasures.

Keywords: SFTS; Immunopathology; Viral pathogenesis.


#Influenza #Hemagglutinins #H2, #H5, #H6, and #H11 are not Targets of Pulmonary #Surfactant Protein D: N-glycan subtypes in host-pathogen interactions (J Virol., abstract)

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

Influenza Hemagglutinins H2, H5, H6, and H11 are not Targets of Pulmonary Surfactant Protein D: N-glycan subtypes in host-pathogen interactions

Lisa Parsons, Yanming An, Li Qi, Mitchell White, Roosmarijn van der Woude, Kevan Hartshorn, Jeffery K. Taubenberger, Robert P. de Vries, John F. Cipollo

DOI: 10.1128/JVI.01951-19



Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low pathogenicity A type influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortmant and emergence in human populations. Since the presence of head region high mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here we investigate the activities of two recombinant human SP-D forms against representative LPAIV including H2N1, H5N1, H6N1, H11N9, an avian H3N8 and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPIAV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite “N165” (H3 numbering) is occupied by high mannose glycans in H3 HA but by complex glycans in all LVIAV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high mannose glycan on the head region our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three dimensional structural analysis.



Low pathogenicity A type influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations as was seen in the 1957 pandemic, where an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first line innate immunity defence, removes IAV through interaction with hemagglutinin (HA) head region high mannose glycan(s). While it is known that both H1 and H3 HAs, have a key high mannose glycosite(s) in the head region, little is known about such glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high mannose glycans, do not interact with SP-D, and that sequence analysis can predict glycan subtype thus predicting presence or absence of this virulence marker.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Avian Influenza; Influenza A; Reassortant strains; H1N1; H2N2; H2N1; H3N2; H3N8; H5N1; H6N1; H11N9; Viral pathogenesis.


Expression of 9-O- and 7,9-O-Acetyl Modified #Sialic Acid in Cells and Their Effects on #Influenza Viruses (MBio, abstract)

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

Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Karen N. Barnard, Brian R. Wasik, Justin R. LaClair, David W. Buchholz, Wendy S. Weichert, Brynn K. Alford-Lawrence, Hector C. Aguilar, Colin R. Parrish

Xiang-Jin Meng, Editor

DOI: 10.1128/mBio.02490-19



Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.



Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.

Keywords: Influenza A; Influenza B; Influenza C; Influenza D; Viral pathogenesis.