14-Deoxy-11,12-didehydroandrographolide inhibits apoptosis in #influenza A(#H5N1) virus-infected #human #lung epithelial #cells via the caspase-9-dependent intrinsic apoptotic pathway which contributes to its antiviral activity (Antiviral Res., abstract)

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

Antiviral Research | Available online 20 July 2020, 104885 | In Press, Journal Pre-proof

14-Deoxy-11,12-didehydroandrographolide inhibits apoptosis in influenza A(H5N1) virus-infected human lung epithelial cells via the caspase-9-dependent intrinsic apoptotic pathway which contributes to its antiviral activity

Wentao Cai a, Haimei Wen a, Qinyang Zhou a, Lei Wu a, Yong Chen a, Hongbo Zhou b, Meilin Jin b

a Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, National & Local Joint Engineering Research Center of High-throughput Drug Screening Technology, State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, 430062, China; b State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, The Cooperative Innovation Center for Sustainable Pig Production, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China

Received 6 March 2020, Revised 26 May 2020, Accepted 22 June 2020, Available online 20 July 2020.

DOI: https://doi.org/10.1016/j.antiviral.2020.104885



  • DAP inhibits caspase-3 and caspase-9 activation induced by influenza A(H5N1) virus in vitro.
  • DAP reduces mitochondrial membrane potential loss and represses cytochrome crelease.
  • DAP exerts anti-apoptotic effect via the caspase-9-dependent intrinsic pathway.
  • Anti-H5N1 ability of DAP partly depends on the inhibition of caspase-9 activity.
  • We propose that DAP is a potential candidate of anti-influenza drug.



Influenza A virus (IAV) infection represents a global health challenge. Excavating antiviral active components from traditional Chinese medicine (TCM) is a promising anti-IAV strategy. Our previous studies have demonstrated that 14-deoxy-11,12-didehydroandrographolide (DAP), a major ingredient of a TCM herb called Andrographis paniculata, shows anti-IAV activity that is mainly effective against A/chicken/Hubei/327/2004 (H5N1), A/duck/Hubei/XN/2007 (H5N1), and A/PR/8/34 (H1N1) in vitro and in vivo. However, the underlying anti-IAV molecular mechanism of DAP needs further investigation. In the present work, we found that DAP can significantly inhibit the apoptosis of human lung epithelial (A549) cells infected with A/chicken/Hubei/327/2004 (H5N1). After DAP treatment, the protein expression levels of cleaved PARP, cleaved caspase-3, and cleaved caspase-9, and the activities of caspase-3 and caspase-9 in H5N1-infected A549 cells were all obviously downregulated. However, DAP had no inhibitory effect on caspase-8 activity and cleaved caspase-8 production. Meanwhile, the efficacy of DAP in reducing the apoptotic cells was lost after using the inhibitor of caspase-3 or caspase-9 but remained intact after the caspase-8 inhibitor treatment. Moreover, DAP efficiently attenuated the dissipation of mitochondrial membrane potential, suppressed cytochrome c release from the mitochondria to the cytosol, and decreased the protein expression ratio of Bax/Bcl-2 in the mitochondrial fraction. Furthermore, the silencing of caspase-9 reduced the yield of nucleoprotein (NP) and disabled the inhibitory ability of DAP in NP production in A549 cells. Overall results suggest that DAP exerts its antiviral effects by inhibiting H5N1-induced apoptosis on the caspase-9-dependent intrinsic/mitochondrial pathway, which may be one of the anti-H5N1 mechanisms of DAP.

© 2020 Elsevier B.V. All rights reserved.

Keywords: A/H5N1; Avian Influenza; Antivirals.


Adjuvanted #H5N1 #influenza #vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans (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.]

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans

Ali H. Ellebedy,  Raffael Nachbagauer, Katherine J. L. Jackson, Ya-Nan Dai, Julianna Han, Wafaa B. Alsoussi, Carl W. Davis, Daniel Stadlbauer, Nadine Rouphael, Veronika Chromikova, Megan McCausland, Cathy Y. Chang, Mario Cortese, Mary Bower, Chakravarthy Chennareddy, Aaron J. Schmitz, Veronika I. Zarnitsyna, Lilin Lai, Arvind Rajabhathor, Cheyann Kazemian, Rustom Antia, Mark J. Mulligan,  Andrew B. Ward,  Daved H. Fremont, Scott D. Boyd, Bali Pulendran, Florian Krammer, and Rafi Ahmed

PNAS first published July 13, 2020 https://doi.org/10.1073/pnas.1906613117

Contributed by Rafi Ahmed, November 8, 2019 (sent for review April 19, 2019; reviewed by Robert L. Coffman and Marc K. Jenkins)



The development of a universal influenza vaccine is a major public health need globally, and identifying the optimal formulation will be an important first step for developing such a vaccine. Here we show that a two-dose immunization of humans with an inactivated, AS03-adjuvanted H5N1 avian influenza virus vaccine engaged both the preexisting memory and naive B cell compartments. Importantly, we show that the recruited memory B cells after first immunization were directed against conserved epitopes within the H5 HA stem region while the responses after the second immunization were mostly directed against strain-specific epitopes within the HA globular head. Taken together these findings have broad implications toward optimizing vaccination strategies for developing more effective vaccines against pandemic viruses.



There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.



1 Present address: Division of Immunobiology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.

2 Present address: Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.

3 Present address: Departments of Pathology, and Microbiology & Immunology, Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA 94305.

4 Present address: Division of Infectious Diseases and Immunology, Department of Medicine, New York University, New York, NY 10016.

5 To whom correspondence may be addressed. Email: rahmed@emory.edu.

Author contributions: A.H.E. and R. Ahmed designed research; A.H.E., R.N., Y.-N.D., J.H., W.B.A., D.S., N.R., V.C., M.M., C.Y.C., and C.K. performed research; K.J.L.J., M.C., M.B., C.C., A.J.S., L.L., A.R., M.J.M., A.B.W., D.H.F., S.D.B., B.P., and F.K. contributed new reagents/analytic tools; A.H.E., K.J.L.J., C.W.D., V.I.Z., R. Antia, D.H.F., and S.D.B. analyzed data; and A.H.E. and R. Ahmed wrote the paper.

Reviewers: R.L.C., University of California; and M.K.J., University of Minnesota.

The authors declare no competing interest.

Data deposition: Structures have been deposited in the Electron Microscopy Data Bank (accession codes: 1F03: EMD-20570 1H09: EMD-20571 1C01: EMD-20569) and BioProject Sequence Read Archive (accession no. PRJNA533650).

This article contains supporting information online  at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1906613117/-/DCSupplemental. Published under the PNAS license.

Keywords: Avian Influenza; H5N1; Vaccines.


The possible #impairment of #respiratory‐related #neural #loops may be associated with the #silent #pneumonia induced by #SARS‐CoV‐2 (J Med Virol., abstract)

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

The possible impairment of respiratory‐related neural loops may be associated with the silent pneumonia induced by SARS‐CoV‐2

Bai‐Hong Tan,  Yan Zhang,  Yue Gui,  Shuang Wu,  Yan‐Chao Li

First published: 11 June 2020 | DOI:  https://doi.org/10.1002/jmv.26158

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jmv.26158



As compared to many other viral pulmonary infections, there existed several peculiar manifestations in the COVID‐19 patients, including the “silence” of pneumonia in both mild and severe cases and a long intensive care unit stay for those requiring invasive mechanical ventilation. Similar silent pneumonia has been documented in the infection induced by H5N1 influenza virus HK483, and was found to result from the direct attack of the virus on the bronchopulmonary C‐fibers at the early stage and the final infection in the brainstem at the late stage. The long stay of critical patients in the intensive care unit is possibly due to the depression of central respiratory drive, which resulted in the failure to wean from the mechanic ventilation. Carotid and aortic bodies and bronchopulmonary C‐fibers are two key peripheral components responsible for the chemosensitive responses in the respiratory system, while triggering respiratory reflexes depends predominantly on the putative chemosensitive neurons located in the pontomedullary nuclei. In view of the findings for H5N1 influenza virus, the silence of pneumonia induced by SARS‐CoV‐2 may be due to the possible impairment of peripheral chemosensitive reflexes as well as the damage to the respiratory‐related central neurons. (195 words)

This article is protected by copyright. All rights reserved.

Keywords: SARS-CoV-2; COVID-19; Avian Influenza; H5N1; Neuroinvasion; Neurology.


#Aminoacid #Substitutions in #Antigenic #Region B of #Hemagglutinin Play a Critical Role in the #Antigenic #Drift of Subclade Highly Pathogenic #H5NX #Influenza Viruses (Transbound Emerg Dis., abstract)

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

 Transbound Emerg Dis. 2020 Jan;67(1):263-275. doi: 10.1111/tbed.13347. Epub 2019 Sep 28.

Amino Acid Substitutions in Antigenic Region B of Hemagglutinin Play a Critical Role in the Antigenic Drift of Subclade Highly Pathogenic H5NX Influenza Viruses

Juan Li 1 2, Min Gu 1 3 4, Kaituo Liu 1, Ruyi Gao 1, Wenqiang Sun 1, Dong Liu 1, Kaijun Jiang 1, Lei Zhong 1, Xiaoquan Wang 1 3 4, Jiao Hu 1 3 4, Shunlin Hu 1 3 4, Xiaowen Liu 1 3 4, Weifeng Shi 2, Hongguang Ren 5, Daxin Peng 1 3 4, Xinan Jiao 1 3 4, Xiufan Liu 1 3 4

Affiliations: 1 Key Laboratory of Animal Infectious Diseases, Ministry of Agriculture, College of Veterinary Medicine, Yangzhou University, Yangzhou, China. 2 Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Universities of Shandong, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China. 3 Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China. 4 Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture, Yangzhou University, Yangzhou, China. 5 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing, China.

PMID: 31484213 DOI: 10.1111/tbed.13347



As one of the important control strategies for highly pathogenic avian influenza (HPAI) in China, vaccination has been implemented compulsively in poultry flocks since 2004. However, the emergence and dominance of the circulating antigenic variants require the update of vaccines periodically. In order to investigate the key molecular sites responsible for the antigenic drift, a total of 13 amino acid positions divergent between clade 2.3.4 H5 viruses and their descendent subclade variants in or around the recognized antigenic epitopes A-E were initially identified through inspecting a comprehensive HA sequence alignment of the H5 subtype HPAI viruses. Subsequently, a panel of single-site or multi-site HA mutants was constructed by reverse genetics with two H5N1 viruses of S (clade 2.3.4) and QD1 (subclade as the HA backbone to study their antigenic variations, respectively. The hemagglutination-inhibition assay revealed an evident impact of mutations at sites 88, 156, 205, 208, 239 and 289 to the HA antigenicity and highlighted that the amino acid substitutions located in the antigenic region B, especially the combined mutations at sites 205 and 208, were the major antigenic determinant which was also consistent with results from flow cytometry and antigenic mapping. Our findings provided more insights into the molecular mechanism of antigenic drift of the H5 subtype HPAI virus, which would be helpful for the selection of vaccine candidates and accordingly for the prevention and control of this devastating viral agent.

Keywords: Highly pathogenic avian influenza; antigenic drift; clade; hemagglutinin (HA) gene.

© 2019 Blackwell Verlag GmbH.

Keywords: Avian Influenza; Poultry; China; H5N1.


#Tropism, #replication competence, and innate immune responses of the #coronavirus #SARS-CoV-2 in #human #respiratory tract and #conjunctiva: an analysis in ex-vivo and in-vitro cultures (Lancet Resp Med., abstract)

[Source: The Lancet Respiratory Medicine, full page: (LINK). Abstract, edited.]

Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures

Kenrie P Y Hui, PhD, Man-Chun Cheung, MSc, Ranawaka A P M Perera, PhD, Ka-Chun Ng, BSc, Christine H T Bui, PhD, John C W Ho, PhD, Mandy M T Ng, BSc, Denise I T Kuok, PhD, Kendrick C Shih, MBBS, Prof Sai-Wah Tsao, PhD, Prof Leo L M Poon, DPhil, Prof Malik Peiris, FRCPath, Prof John M Nicholls, FRCPA, Michael C W Chan, PhD

Published: May 07, 2020 | DOI: https://doi.org/10.1016/S2213-2600(20)30193-4




Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, causing a respiratory disease (coronavirus disease 2019, COVID-19) of varying severity in Wuhan, China, and subsequently leading to a pandemic. The transmissibility and pathogenesis of SARS-CoV-2 remain poorly understood. We evaluate its tissue and cellular tropism in human respiratory tract, conjunctiva, and innate immune responses in comparison with other coronavirus and influenza virus to provide insights into COVID-19 pathogenesis.


We isolated SARS-CoV-2 from a patient with confirmed COVID-19, and compared virus tropism and replication competence with SARS-CoV, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and 2009 pandemic influenza H1N1 (H1N1pdm) in ex-vivo cultures of human bronchus (n=5) and lung (n=4). We assessed extrapulmonary infection using ex-vivo cultures of human conjunctiva (n=3) and in-vitro cultures of human colorectal adenocarcinoma cell lines. Innate immune responses and angiotensin-converting enzyme 2 expression were investigated in human alveolar epithelial cells and macrophages. In-vitro studies included the highly pathogenic avian influenza H5N1 virus (H5N1) and mock-infected cells as controls.


SARS-CoV-2 infected ciliated, mucus-secreting, and club cells of bronchial epithelium, type 1 pneumocytes in the lung, and the conjunctival mucosa. In the bronchus, SARS-CoV-2 replication competence was similar to MERS-CoV, and higher than SARS-CoV, but lower than H1N1pdm. In the lung, SARS-CoV-2 replication was similar to SARS-CoV and H1N1pdm, but was lower than MERS-CoV. In conjunctiva, SARS-CoV-2 replication was greater than SARS-CoV. SARS-CoV-2 was a less potent inducer of proinflammatory cytokines than H5N1, H1N1pdm, or MERS-CoV.


The conjunctival epithelium and conducting airways appear to be potential portals of infection for SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated similarly in the alveolar epithelium; SARS-CoV-2 replicated more extensively in the bronchus than SARS-CoV. These findings provide important insights into the transmissibility and pathogenesis of SARS-CoV-2 infection and differences with other respiratory pathogens.


US National Institute of Allergy and Infectious Diseases, University Grants Committee of Hong Kong Special Administrative Region, China; Health and Medical Research Fund, Food and Health Bureau, Government of Hong Kong Special Administrative Region, China.

Keywords: SARS-CoV; SARS-CoV-2; MERS-CoV; H5N1; H1N1pdm09; Cytokines; Immunopathology.


#Phenotypic effects of #substitutions within the #receptor binding site of highly pathogenic #avian #influenza #H5N1 observed during #human #infection (J Virol., abstract)

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

Phenotypic effects of substitutions within the receptor binding site of highly pathogenic avian influenza H5N1 observed during human infection.

Dirk Eggink, Monique Spronken, Roosmarijn van der Woude, Jocynthe Buzink, Frederik Broszeit, Ryan McBride, Hana A Pawestri, Vivi Setiawaty, James C. Paulson, Geert-Jan Boons, Ron A.M. Fouchier, Colin A. Russell, Menno D. de Jong, Robert P. de Vries

DOI: 10.1128/JVI.00195-20



Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution towards a transmittable virus.

Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next generation sequencing. Here we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the RBS resulted in viable virus, but virus replication, entry and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans.



H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase HA stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability and receptor specificity. None of the tested substitutions resulted in a clear step towards a human adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

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

Keywords: Avian Influenza; H5N1; Human.


#Comparison of #Pathological Changes and #Pathogenic Mechanisms Caused by #H1N1 Influenza Virus, HPAI #H5N1, #SARS-CoV, #MERS-CoV and 2019-nCoV #Coronavirus (Zhonghua Bing Li Xue Za Zhi, abstract)

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

Zhonghua Bing Li Xue Za Zhi, 40 (0), E006 2020 Mar 16 [Online ahead of print]

[Comparison of Pathological Changes and Pathogenic Mechanisms Caused by H1N1 Influenza Virus, Highly Pathogenic H5N1 Avian Influenza Virus, SARS-CoV, MERS-CoV and 2019-nCoV Coronavirus]

[Article in Chinese]

M Liu 1, R E Feng 2, Q Li 3, H K Zhang 1, Y G Wang 1

Affiliations: 1 Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China. 2 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. 3 Shunyi Hospital of Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 101300, China.

PMID: 32174094 DOI: 10.3760/cma.j.cn112151-20200301-00155



The rapid development of the new coronavirus pneumonia epidemic in Wuhan, China, has caused severe impact on the country, but so far, little is known about the pathological changes and pathogenesis of the new coronavirus pneumonia. This article summarizes the pathological changes of severe influenza virus H1N1, highly pathogenic avian influenza virus H5N1, SARS-CoV, MERS-CoV, and 2019-nCoV coronavirus that cause major outbreaks of viral infectious diseases. The autopsy lung tissues are diffuse. Alveolar damage (DAD), but pathological manifestations caused by different viruses are different. Severe influenza virus 2009 H1N1 virus binds to receptors α-2,6-SA and α-2,3-SA, except for DAD lesions It is often accompanied by inflammatory lesions of the upper respiratory tract, trachea, bronchi and bronchioles, and is more likely to be complicated by bacterial infection. The highly pathogenic avian influenza virus H5N1 mainly binds α-2,3-SA receptors, mainly involving alveolar epithelium and bronchioles. Rarely, upper respiratory tract and trachea and bronchial lesions are often associated with focal pulmonary hemorrhage and lung tissue necrosis. Mechanization and fibrosis are rare. SARS-CoV enters cells by binding to angiotensin-converting enzyme 2 (ACE2), and the lesions are related to the course of disease. The DAD exudation period is generally seen in patients who die within 10 to 14 days. Patients with a disease course of more than 10 days showed mechanized DAD, often accompanied by occlusive bronchiolitis with organic pneumonia-like changes and significant multinucleated giant cells in the alveolar cavity. In patients with SARS-CoV and H5N1 infection, lymphocyte depletion in the spleen and lymph nodes, acute tubular necrosis, and hemophagocytic cells in the bone marrow were seen in the extrapulmonary organs.

Keywords: SARS-CoV-2; SARS-CoV; H1N1pdm09; H5N1.


#Avian #influenza #human #infections at the human-animal interface (J Infect Dis., abstract)

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

Avian influenza human infections at the human-animal interface

Damien A M Philippon, Peng Wu, Benjamin J Cowling, Eric H Y Lau

The Journal of Infectious Diseases, jiaa105, https://doi.org/10.1093/infdis/jiaa105

Published: 10 March 2020




Avian influenza A viruses (AIVs) are among the most concerning emerging and re-emerging pathogens because of the potential risk in causing an influenza pandemic with catastrophic impact. The recent increase in domestic animals and poultry worldwide was followed by an increase of human AIV outbreaks reported.


We reviewed the epidemiology of human infections with AIV from the literature including reports from the World Health Organization, extracting information on virus subtype, time, location, age, sex, outcome and exposure.


We described the characteristics of more than 2,500 laboratory-confirmed human infections with AIVs. Human infections with H5N1 and H7N9 were more frequently reported than other subtypes. The risk of death was highest among reported cases infected with H5N1, H5N6, H7N9 and H10N8 infections. Older people and males tended to have a lower risk of infection with most AIV subtypes, except for H7N9. Visiting live poultry markets were mostly reported by H7N9, H5N6 and H10N8 cases, while exposure to sick or dead bird mostly reported by H5N1, H7N2, H7N3, H7N4, H7N7 and H10N7 cases.


Understanding the profile of human cases of different AIV subtypes would guide control strategy. Continued monitoring of human infections with AIVs is essential for pandemic preparedness.

avian influenza, human infection, review

Issue Section:  Review

This content is only available as a PDF.

© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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: Avian Influenza; Human; H5N1; H5N6; H7N3; H7N7; H7N9; H9N2; H10N8.


Quantifying within-host #diversity of #H5N1 #influenza viruses in #humans and #poultry in #Cambodia (PLOS Pathog., abstract)

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


Quantifying within-host diversity of H5N1 influenza viruses in humans and poultry in Cambodia

Louise H. Moncla , Trevor Bedford, Philippe Dussart, Srey Viseth Horm, Sareth Rith, Philippe Buchy, Erik A. Karlsson, Lifeng Li, Yongmei Liu, Huachen Zhu, Yi Guan, Thomas C. Friedrich, Paul F. Horwood


Published: January 17, 2020 / DOI: https://doi.org/10.1371/journal.ppat.1008191 / This is an uncorrected proof.



Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.


Author summary

H5N1 avian influenza viruses can cross species barriers and cause severe disease in humans. H5N1 viruses currently cannot replicate and transmit efficiently among humans, but animal infection studies and modeling experiments have suggested that human adaptation may require only a few mutations. However, data from natural spillover infection has been limited, posing a challenge for risk assessment. Here, we analyze a unique dataset of deep sequence data from H5N1 virus-infected humans and domestic ducks in Cambodia. We find that well-known markers of human receptor binding and replication arise in multiple, independent humans. We also find that 3 mutations detected within-host are enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adapting. However, we also show that within-host evolution in both humans and ducks are shaped heavily by purifying selection and genetic drift, and that a large fraction of within-host variation is never detected on the H5N1 phylogeny. Taken together, our data show that H5N1 viruses do generate human-adapting mutations during natural infection. However, short infection times, purifying selection, and genetic drift may severely limit how much H5N1 viruses can evolve during the course of a single infection.


Citation: Moncla LH, Bedford T, Dussart P, Horm SV, Rith S, Buchy P, et al. (2020) Quantifying within-host diversity of H5N1 influenza viruses in humans and poultry in Cambodia. PLoS Pathog 16(1): e1008191. https://doi.org/10.1371/journal.ppat.1008191

Editor: Wendy S. Barclay, Imperial College London, UNITED KINGDOM

Received: July 8, 2019; Accepted: November 4, 2019; Published: January 17, 2020

Copyright: © 2020 Moncla 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 raw sequence data are available in the SRA under accession number PRJNA547644 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA547644). All code used to analyze the data, as well as data files with within-host variant calls and phylogenetic trees are available at https://github.com/blab/h5n1-cambodia.

Funding: The study was funded by the US Agency for International Development (grant No. AID-442-G-14-00005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Dr. Philippe Buchy is a former Head of Virology at Institut Pasteur du Cambodge and is currently an employee of GSK Vaccines, Singapore. The other authors declare no conflict of interest.

Keywords: Avian Influenza, H5N1, Human, Poultry, Cambodia.


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.