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.]

OPEN ACCESS /  PEER-REVIEWED / RESEARCH ARTICLE

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

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

 

Abstract

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.

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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.

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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.

 

Abstract

BACKGROUND & OBJECTIVES:

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.

METHODS:

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.

RESULTS:

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.

INTERPRETATION & CONCLUSIONS:

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.

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Comparative #Pathogenicity and #Transmissibility of #H1N1pdm09, #Avian #H5N1, and #Human #H7N9 #Influenza Viruses in Tree #Shrews (Front Microbiol., abstract)

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

Front Microbiol. 2019 Dec 20;10:2955. doi: 10.3389/fmicb.2019.02955. eCollection 2019.

Comparative Pathogenicity and Transmissibility of Pandemic H1N1, Avian H5N1, and Human H7N9 Influenza Viruses in Tree Shrews.

Xu S1, Li X1, Yang J1, Wang Z1, Jia Y1, Han L1, Wang L1, Zhu Q1.

Author information: 1 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.

 

Abstract

Influenza A viruses (IAVs) continuously challenge the poultry industry and human health. Studies of IAVs are still hampered by the availability of suitable animal models. Chinese tree shrews (Tupaia belangeri chinensis) are closely related to primates physiologically and genetically, which make them a potential animal model for human diseases. In this study, we comprehensively evaluated infectivity and transmissibility in Chinese tree shrews by using pandemic H1N1 (A/Sichuan/1/2009, pdmH1N1), avian-origin H5N1 (A/Chicken/Gansu/2/2012, H5N1) and early human-origin H7N9 (A/Suzhou/SZ19/2014, H7N9) IAVs. We found that these viruses replicated efficiently in primary tree shrew cells and tree shrews without prior adaption. Pathological lesions in the lungs of the infected tree shrews were severe on day 3 post-inoculation, although clinic symptoms were self-limiting. The pdmH1N1 and H7N9 viruses, but not the H5N1 virus, transmitted among tree shrews by direct contact. Interestingly, we also observed that unadapted H7N9 virus could transmit from tree shrews to naïve guinea pigs. Virus-inoculated tree shrews generated a strong humoral immune response and were protected from challenge with homologous virus. Taken together, our findings suggest the Chinese tree shrew would be a useful mammalian model to study the pathogenesis and transmission of IAVs.

Copyright © 2019 Xu, Li, Yang, Wang, Jia, Han, Wang and Zhu.

KEYWORDS: H1N1; H5N1; H7N9; infectivity; transmissibility; tree shrew

PMID: 31921093 PMCID: PMC6933948 DOI: 10.3389/fmicb.2019.02955

Keywords: Influenza A; H7N9; H5N1; H1N1pdm09; Animal models.

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The fit of #codon usage of #human-isolated #avian #influenza A viruses to human (Infect Genet Evol., abstract)

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

Infect Genet Evol. 2020 Jan 6:104181. doi: 10.1016/j.meegid.2020.104181. [Epub ahead of print]

The fit of codon usage of human-isolated avian influenza A viruses to human.

Luo W1, Tian L2, Gan Y1, Chen E1, Shen X1, Pan J1, Irwin DM3, Chen RA4, Shen Y5.

Author information: 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China. 2 Guangdong Provincial Hospital of Chinese Medicine, Zhuhai 519015, China. 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada; Banting and Best Diabetes Centre, University of Toronto, Toronto M5S 1A8, Canada. 4 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; Zhaoqing Institute of Biotechnology, Zhaoqing 526238, China. Electronic address: chensa727@126.com. 5 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; Zhaoqing Institute of Biotechnology, Zhaoqing 526238, China. Electronic address: shenyy@scau.edu.cn.

 

Abstract

Avian influenza A viruses (AIVs) classify into 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes. Even though H1N1 and H3N2 subtypes usually circulate among humans leading to infection, occasionally, H5, H6, H7, H9, and H10 that circulate in poultry also infect humans, and especially H5N1 and H7N9. Efficient virus replication is a critical factor that influences infection. Codon usage of a virus must coevolve with its host for efficient viral replication, therefore, we conduct a comprehensive analysis of codon usage bias in human-isolated AIVs to test their adaptation to host expression system. The relative synonymous codon usage (RSCU) pattern, and the codon adaptation index (CAI) are calculated for this purpose. We find that all human-isolated AIVs tend to eliminate GC and CpG compositions, which may prevent activation of the host innate immune system. Although codon usage differs between AIV subtypes, our data support the conclusion that natural selection has played a major role and mutation pressure a minor role in shaping codon usage bias in all AIVs. Our efforts discover that codon usage of genes encoding surface proteins of H5N1, and the polymerase genes of H7N9 has better fit to the human expression system. This may associate with their better replication and infection in human.

Copyright © 2019. Published by Elsevier B.V.

KEYWORDS: Adaptation; Avian influenza A viruses; Codon usage; H5N1; H7N9

PMID: 31918040 DOI: 10.1016/j.meegid.2020.104181

Keywords: Avian Influenza; H5N1; H7N9; Human; Evolution.

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Evaluation of #potential #risk of #transmission of #avian #influenza A viruses at live #bird #markets in response to unusual #crow die-offs in #Bangladesh (Influenza Other Respir Viruses, abstract)

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

Influenza Other Respir Viruses. 2020 Jan 7. doi: 10.1111/irv.12716. [Epub ahead of print]

Evaluation of potential risk of transmission of avian influenza A viruses at live bird markets in response to unusual crow die-offs in Bangladesh.

Rahman M1,2, Mangtani P3, Uyeki TM4, Cardwell JM1, Torremorell M5, Islam A6, Samad MA7, Muraduzzaman AKM2, Giasuddin M7, Sarkar S2, Alamgir ASM2, Salimuzzaman M2, Flora MS2.

Author information: 1 Royal Veterinary College, Hatfield, UK. 2 Institute of Epidemiology, Disease Control and Research, Dhaka, Bangladesh. 3 London School of Hygiene and Tropical Medicine, London, UK. 4 Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA. 5 University of Minnesota, Twin Cities, MN, USA. 6 EcoHealth Alliance, New York, NY, USA. 7 Bangladesh Livestock Research Institute (BLRI), Savar, Bangladesh.

 

Abstract

In response to unusual crow die-offs from avian influenza A(H5N1) virus infection during January-February 2017 in Dhaka, Bangladesh, a One Health team assessed potential infection risks in live bird markets (LBMs). Evidence of aerosolized avian influenza A viruses was detected in LBMs and in the respiratory tracts of market workers, indicating exposure and potential for infection. This study highlighted the importance of surveillance platforms with a coordinated One Health strategy to investigate and mitigate zoonotic risk.

© 2020 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

KEYWORDS: Bangladesh; avian influenza; avian influenza A virus; influenza in birds; live bird market; pathogen transmission

PMID: 31912608 DOI: 10.1111/irv.12716

Keywords: Avian Influenza; H5N1; Poultry; Live poultry markets; Bangladesh.

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#Biochemistry and computer generated graph comparison of the #structural and nonstructural #proteins of #spanish-1918 #Influenza, pandemic-2009, and #birdflu viruses (Acta Biochem Pol., abstract)

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

Acta Biochim Pol. 2019 Jul 24;66(3):329-336. doi: 10.18388/abp.2019_2795.

Biochemistry and computer generated graph comparison of the structural and nonstructural proteins of spanish-1918 Influenza, pandemic-2009, and bird flu viruses.

Mahardika GN1, Suartha NI2, Kencana GAY1, Suardana IBK1, Mahardika WW3, Budayanti NS4.

Author information: 1 Virology Laboratory, Faculty of Veterinary Medicine Udayana University, Denpasar, Bali, Indonesia. 2 Animal Hospital, Faculty of Veterinary Medicine Udayana University, Denpasar, Bali, Indonesia. 3 Faculty of Visual Communication Design of Bina Nusantara University, Tanggerang-Banten, Indonesia. 4 Microbiology Department, Faculty of Medicine Udayana University, Denpasar, Bali, Indonesia.

 

Abstract

The potential emergence of deadly pandemic influenza viruses is unpredictable and most have emerged with no forewarning. The distinct epidemiological and pathological patterns of the Spanish (H1N1), pandemic-2009 (H1N1), and avian influenza (H5N1), known as bird flu, viruses may allow us to develop a ‘template’ for possible emergence of devastating pandemic strains. Here, we provide a detailed molecular dissection of the structural and nonstructural proteins of this triad of viruses. GenBank data for three representative strains were analyzed to determine the polymorphic amino acids, genetic distances, and isoelectric points, hydrophobicity plot, and protein modeling of various proteins. We propose that the most devastating pandemic strains may have full-length PB1-F2 protein with unique residues, highly cleavable HA, and a basic NS1. Any newly emerging strain should be compared with these three strains, so that resources can be directed appropriately.

PMID: 31531422 DOI: 10.18388/abp.2019_2795 [Indexed for MEDLINE] Free full text

Keywords: Avian Influenza; Influenza A; Pandemic Influenza; H1N1; H1N1pdm09; H5N1.

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The #PB2 #Polymerase Host Adaptation Substitutions Prime #Avian #Indonesia Sub Clade 2.1 #H5N1 Viruses for Infecting #Humans (Viruses, abstract)

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

Viruses. 2019 Mar 22;11(3). pii: E292. doi: 10.3390/v11030292.

The PB2 Polymerase Host Adaptation Substitutions Prime Avian Indonesia Sub Clade 2.1 H5N1 Viruses for Infecting Humans.

Wang P1, Song W2,3, Mok BW4, Zheng M5, Lau SY6, Liu S7, Chen P8, Huang X9, Liu H10, Cremin CJ11, Chen H12.

Author information: 1 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. puiwang@hku.hk. 2 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. wjsong@hku.hk. 3 State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, Guangzhou Medical University, Guangzhou 510180, China. wjsong@hku.hk. 4 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. bobomok@hku.hk. 5 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. min.zheng@stjude.org. 6 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. sylau926@hku.hk. 7 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. siwen531@CONNECT.HKU.HK. 8 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. u3508816@connect.hku.hk. 9 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. stevehxf@connect.hku.hk. 10 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. lhlotus@connect.hku.hk. 11 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. conor93@connect.hku.hk. 12 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China. hlchen@hku.hk.

 

Abstract

Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2⁻3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection.

KEYWORDS: H5N1; Influenza virus; PB2; RNP; cross species transmission; host adaptation

PMID: 30909490 PMCID: PMC6480796 DOI: 10.3390/v11030292 [Indexed for MEDLINE] Free PMC Article

Keywords: Avian Influenza; H5N1; Human; Poultry; Indonesia, Animal models.

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