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Helin, A. S., Wille, M., Atterby, C., Jarhult, J. D., Waldenström, J. & Chapman, J. R. (2018). A rapid and transient innate immune response to avian influenza infection in mallards. Molecular Immunology, 95, 64-72
Open this publication in new window or tab >>A rapid and transient innate immune response to avian influenza infection in mallards
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2018 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 95, p. 64-72Article in journal (Refereed) Published
Abstract [en]

The vertebrate innate immune system provides hosts with a rapid, non-specific response to a wide range of invading pathogens. However, the speed and duration of innate responses will be influenced by the co-evolutionary dynamics of specific host-pathogen combinations. Here, we show that low pathogenic avian influenza virus (LPAI) subtype H1N1 elicits a strong but extremely transient innate immune response in its main wildlife reservoir, the mallard (Anas platyrhynchos). Using a series of experimental and methodological improvements over previous studies, we followed the expression of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistance gene (Mx) in mallards semi-naturally infected with low pathogenic H1N1. One day post infection, both RIG-I and Mx were significantly upregulated in all investigated tissues. By two days post infection, the expression of both genes had generally returned to basal levels, and remained so for the remainder of the experiment. This is despite the fact that birds continued to actively shed viral particles throughout the study period. We additionally show that the spleen plays a particularly active role in the innate immune response to LPAI. Waterfowl and avian influenza viruses have a long co-evolutionary history, suggesting that the mallard innate immune response has evolved to provide a minimum effective response to LPAIs such that the viral infection is brought under control while minimising the damaging effects of a sustained immune response.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Anas platyrhynchos, Zoonotic disease, Avian disease, Gene expression, Retinoic acid inducible gene 1 RIG-I, Myxovirus resistance gene Mx, Influenza A virus, LPAI, HPAI, Ecoimmunology, Tolerance
National Category
Immunology
Research subject
Ecology, Zoonotic Ecology
Identifiers
urn:nbn:se:lnu:diva-72297 (URN)10.1016/j.molimm.2018.01.012 (DOI)000428096900008 ()29407578 (PubMedID)2-s2.0-85041644274 (Scopus ID)
Available from: 2018-04-06 Created: 2018-04-06 Last updated: 2020-02-07Bibliographically approved
Helin, A. S., Aarts, L., Bususu, I., Andersson, H. S., Rosengren, J., Chapman, J. R. & Waldenström, J. (2018). Antimicrobial differences between AvBDs in mallards (Anas platyrhynchos). In: : . Paper presented at IMAP 2018: 8th International Meeting on Antimicrobial Peptides, Sep 2-4, 2018. Edinburgh, Scotland..
Open this publication in new window or tab >>Antimicrobial differences between AvBDs in mallards (Anas platyrhynchos)
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2018 (English)Conference paper, Poster (with or without abstract) (Other academic)
National Category
Evolutionary Biology
Research subject
Ecology, Evolutionary Biology
Identifiers
urn:nbn:se:lnu:diva-77430 (URN)
Conference
IMAP 2018: 8th International Meeting on Antimicrobial Peptides, Sep 2-4, 2018. Edinburgh, Scotland.
Note

Ej belagd 20190222

Available from: 2018-08-28 Created: 2018-08-28 Last updated: 2019-02-22Bibliographically approved
Helin, A. S., Wille, M., Atterby, C., Jarhult, J., Waldenström, J. & Chapman, J. R. (2018). Expression of immune genes RIG-I and Mx in mallard ducks infected with low pathogenic avian influenza (LPAI): A dataset. Data in Brief, 18, 1562-1566
Open this publication in new window or tab >>Expression of immune genes RIG-I and Mx in mallard ducks infected with low pathogenic avian influenza (LPAI): A dataset
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2018 (English)In: Data in Brief, E-ISSN 2352-3409, Vol. 18, p. 1562-1566Article in journal (Refereed) Published
Abstract [en]

This article provides data on primer sequences used to amplify the innate immune genes RIG-I and Mx and a set of normalizing reference genes in mallards (Anal platyrhynchos), and shows which reference genes are stable, per tissue, for our experimental settings. Data on the expressional changes of these two genes over a time-course of infection with low pathogenic avian influenza virus (LPAI) are provided. Individual-level data are also presented, including LPAI infection load, and per tissue gene expression of RIG-I and Mx. Gene expression in two outlier individuals is explored in more depth. (C) 2018 The Authors. Published by Elsevier Inc.

Place, publisher, year, edition, pages
Elsevier, 2018
National Category
Immunology
Research subject
Ecology, Zoonotic Ecology
Identifiers
urn:nbn:se:lnu:diva-79497 (URN)10.1016/j.dib.2018.04.061 (DOI)000449815400215 ()29904657 (PubMedID)2-s2.0-85041687141 (Scopus ID)
Available from: 2019-01-15 Created: 2019-01-15 Last updated: 2020-02-07Bibliographically approved
Helin, A. S., Aarts, L., Bususu, I., Andersson, H. S., Rosengren, J., Chapman, J. R. & Waldenström, J. (2017). Antimicrobial activity differences in reduced vs. oxidized AvBD3b peptides in mallards (Anas platyrhynchos).. In: : . Paper presented at 7th International Meeting on Antimicrobia Peptides, 2017, August 25-27, Copenhagen, Denmark.
Open this publication in new window or tab >>Antimicrobial activity differences in reduced vs. oxidized AvBD3b peptides in mallards (Anas platyrhynchos).
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2017 (English)Conference paper, Poster (with or without abstract) (Other academic)
National Category
Evolutionary Biology
Research subject
Natural Science, Ecology
Identifiers
urn:nbn:se:lnu:diva-71389 (URN)
Conference
7th International Meeting on Antimicrobia Peptides, 2017, August 25-27, Copenhagen, Denmark
Available from: 2018-03-05 Created: 2018-03-05 Last updated: 2018-11-02Bibliographically approved
Helin, A. S., Aarts, L., Chapman, J. R., Andersson, H. S. & Waldenström, J. (2017). Bactericidal tests of mallard (Anas plathyrynchos) ß-defensin alleles. In: : . Paper presented at 7th One Health Sweden Scientific Meeting, Kalmar, Sweden, March 22-23, 2017.
Open this publication in new window or tab >>Bactericidal tests of mallard (Anas plathyrynchos) ß-defensin alleles
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2017 (English)Conference paper, Poster (with or without abstract) (Other academic)
National Category
Evolutionary Biology
Research subject
Ecology, Evolutionary Biology
Identifiers
urn:nbn:se:lnu:diva-71388 (URN)
Conference
7th One Health Sweden Scientific Meeting, Kalmar, Sweden, March 22-23, 2017
Note

Ej belagd 20180306

Available from: 2018-03-05 Created: 2018-03-05 Last updated: 2018-11-02Bibliographically approved
Chapman, J. R., Helin, A. S., Wille, M., Atterby, C., Jarhult, J. D., Fridlund, J. & Waldenström, J. (2016). A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos). PLoS ONE, 11(2), Article ID e0149454.
Open this publication in new window or tab >>A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 2, article id e0149454Article in journal (Refereed) Published
Abstract [en]

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard-a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used beta-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. beta-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.

National Category
Ecology
Research subject
Ecology, Zoonotic Ecology
Identifiers
urn:nbn:se:lnu:diva-51591 (URN)10.1371/journal.pone.0149454 (DOI)000371218400085 ()26886224 (PubMedID)2-s2.0-84960902443 (Scopus ID)
Available from: 2016-03-30 Created: 2016-03-30 Last updated: 2020-02-07Bibliographically approved
Chapman, J. R., Hellgren, O., Helin, A. S., Kraus, R. H. S., Cromie, R. L. & Waldenström, J. (2016). The Evolution of Innate Immune Genes: Purifying and Balancing Selection on beta-Defensins in Waterfowl. Molecular biology and evolution, 33(12), 3075-3087
Open this publication in new window or tab >>The Evolution of Innate Immune Genes: Purifying and Balancing Selection on beta-Defensins in Waterfowl
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2016 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 33, no 12, p. 3075-3087Article in journal (Refereed) Published
Abstract [en]

In disease dynamics, high immune gene diversity can confer a selective advantage to hosts in the face of a rapidly evolving and diverse pathogen fauna. This is supported empirically for genes involved in pathogen recognition and signalling. In contrast, effector genes involved in pathogen clearance may be more constrained. beta-Defensins are innate immune effector genes; their main mode of action is via disruption of microbial membranes. Here, five beta-defensin genes were characterized in mallards (Anas platyrhynchos) and other waterfowl; key reservoir species for many zoonotic diseases. All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific beta-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated beta-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of beta-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian beta-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.

Keywords
Antimicrobial peptides, host defense peptides, ecoimmunology, avian immune system, host-pathogen dynamics
National Category
Evolutionary Biology Immunology
Research subject
Ecology, Evolutionary Biology; Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-59004 (URN)10.1093/molbev/msw167 (DOI)000387925300005 ()2-s2.0-85016214902 (Scopus ID)
Available from: 2016-12-15 Created: 2016-12-14 Last updated: 2020-02-07Bibliographically approved
Chapman, J. R. & Waldenström, J. (2015). With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS ONE, 10(11), Article ID e0141853.
Open this publication in new window or tab >>With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, article id e0141853Article, review/survey (Refereed) Published
Abstract [en]

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with beta-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-47712 (URN)10.1371/journal.pone.0141853 (DOI)000364430700041 ()26555275 (PubMedID)2-s2.0-84953315386 (Scopus ID)
Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2018-11-15Bibliographically approved
Helin, A. S., Chapman, J. R., Tolf, C., Andersson, H. S. & Waldenström, J.From genes to function: variation in antimicrobial activity of avian β-defensin peptides from mallards.
Open this publication in new window or tab >>From genes to function: variation in antimicrobial activity of avian β-defensin peptides from mallards
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Avian β-defensins are an important class of antimicrobial peptides in birds. These short cationic peptides are directly involved in the clearance of infections by membrane disruption, but can also act as immunomodulators and chemotactic agents recruiting immune cells. Recent genomic studies have shown the presence of several different avian β-defensin (AvBD) genes across the avian phylogeny, but also significant copy number variation and occurrence of pseudogenes. In mallard (Anas platyrhynchos) and other waterfowl AvBD genes are conserved and seem to be maintained by purifying selection. Due to their relatively simple peptide structure and direct mode of action, AvBDs is a potential tractable system to investigate how small differences in the gene sequence translates into differences in immune function. Here, we used genomic information from three different mallard defensin loci (AvBD4, AvBD10 and AvBD13) and synthesized the linear peptides from the most common allele of each locus, plus two rare alleles from AvBD13 locus and measured their antimicrobial activity against Gram-negative (E. coli and S. Typhimurium) and Gram-positive (S. aureus and M. luteus) bacteria. In these assays, AvBD4 showed the most potent antibacterial activity against both Gram-negative and Gram-positive bacteria, with an IC50 value of 0.48 mM against S. Typhimurium. Among AvBD13 peptides, the less frequently observed AvBD13:2 variant, was most potent, with IC50 value against S. aureus approximately 15 times lower than that of the most common AvBD13:1. Interestingly, AvBD10 had no antibacterial effect on the tested bacteria. Thus, antimicrobial function varied substantially among loci, but also within the AvBD13 locus, suggesting a direct link between genetic variation and immune function variation. Interestingly, results from assays with AvBD4 and AvBD13 seem to indicate that a higher positive net charge in peptides is associated with a more potent antibacterial effect.

National Category
Immunology
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-91974 (URN)
Available from: 2020-02-07 Created: 2020-02-07 Last updated: 2020-02-21Bibliographically approved
Helin, A. S., Chapman, J. R., Tolf, C., Aarts, L., Bususu, I., Rosengren, J., . . . Waldenström, J.Relation between structure and function of three AvBD3b variants from mallard (Anas platyrhynchos).
Open this publication in new window or tab >>Relation between structure and function of three AvBD3b variants from mallard (Anas platyrhynchos)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Defensins are multifunctional antimicrobial peptides expressed in several tissue types and leucocytes as part of the innate immune response against microbes. Based on the three-dimensional structure and disulfide connectivity, vertebrate defensins are subdivided into α-, β-, and θ-defensins. While all three types have been found in mammals, only β-defensins have been identified in birds. Genetic studies have revealed dozens of different avian β-defensin (AvBD) genes in different bird species, as well as allelic variation for different genes. Knowledge of the relation between avian peptide structure features and antimicrobial activity is however limited. In this study, the structure-functional relations of three variants of AvBD3b, a mallard (Anas platyrhynchos) defensin of evolutionary interest, was investigated. Gene alleles encoding two of these peptides, AvBD3b:1 and AvBD3b:2 are common in mallards, whereas AvBD3b:3 occurs rare. These β-defensin peptides were synthesized as linear peptides and subjected to oxidative folding. The three-dimensional structure of AvBD3b:1, including disulfide bond connectivity, was determined using NMR, and those of AvBD3b:2 and AvBD3b:3 respectively, were modelled using AvBD3b:1 as the template. The antimicrobial activities of folded peptides were compared to those of linear peptides. The NMR analysis showed that folded AvBD3b adopts a three-dimensional structure typical for β-defensins, including C-terminal antiparallel β-sheets and disulfide bond organization between six cysteine (C) residues: C6-C34, C13-C28, and C18-C35. Analyses of antimicrobial activity showed that both folded and linear variants of the three peptides inhibited bacterial growth. However, differences in activity were observed, suggesting that folded AvBD3b:3 was the most efficient against both Gram-negative and Gram-positive bacteria. Taken together, these findings provide additional insight into the influence of amino acid sequence variation and three-dimensional structure on the antimicrobial activity of mallard AvBD3b.

National Category
Immunology
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-91970 (URN)
Available from: 2020-02-07 Created: 2020-02-07 Last updated: 2020-02-21Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2849-1094

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