Expression of immune genes RIG-I and Mx in mallard ducks infected with low pathogenic avian influenza (LPAI): A dataset

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 (Anas 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.


Subject area
Biology More specific subject area Immunology Type of data Table, graph, figure How data was acquired Mallards were infected with low pathogenic AIV, and sacrificed over a time-course. RNA was extracted from harvested tissues and gene expression of immune genes and reference genes was analyzed via RT-qPCR on a LightCycler 480 (Roche). Data analysis was performed using qBase þ and GraphPad Prism. Data format Analyzed Experimental factors Ducks were infected with an H1N1 virus. Extracted RNA was treated with DNase.

Experimental features
Infection of mallards was achieved via a semi-natural, contact infection regime. qPCR results were normalized using a panel of reference genes shown to be stable for the experimental conditions under consideration.

Data source location
Infections were performed at the Swedish Veterinary Institute, Uppsala, Sweden. Molecular lab work was conducted at Linnaeus University, Kalmar, Sweden.

Data accessibility
Data are provided with this article

Value of the data
Avian influenza virus (AIV) infection of mallards was achieved via a semi-natural, contact infection route to mimic natural transmission of the virus.
Infection with low pathogenic AIV provides a contrast to most previous studies that used highly pathogenic AIV to study immune gene expression in mallards.
A set of reference genes that had been experimentally validated as stable under the given experimental treatment were used to stabilize RT-qPCR.
A table summarizing the methodology and findings of previous studies of Mx and/or RIG-I expression in AIV infected ducks is provided.

Data
The dataset provided here provides additional information for Helin et al. [1]. In that paper, we show that the innate immune genes retinoic acid-inducible gene-I (RIG-I) and myxovirus resistance gene (Mx) are rapidly yet transiently upregulated after infection with low pathogenic avian influenza virus (LPAI) subtype H1N1. Helin et al. aims to provide a series of methodological improvements over previous analyses of immune gene expression in ducks infected with avian influenza virus (AIV). Table 1 shows that most previous studies have used highly pathogenic avian influenza virus (HPAI), which is rarely detected in wild mallards [2,3]. Additionally, infection in previous studies was achieved via artificial inoculation comprising potentially unnatural viral doses and infection routes. These previous studies have almost exclusively been conducted on domestic Pekin ducks, rather than the main wildlife reservoir for avian influenza, mallard ducks (Anas platrhynchos). Lastly, most previous studies have used a single, non-validated reference gene (often GAPDH) for normalizing gene expression data. This approach leads to potentially misleading interpretation of data [4]. Table 1 Previous studies of RIG-I and Mx gene expression in mallard and Pekin ducks infected with AIV. Only studies using quantitative real-time PCR to assess patterns of gene expression are included. Only results significantly different from controls are listed, and all fold-changes represent upregulation compared to controls (no study found down-regulation of either gene at any time point). EID 50 is 50% egg infectious dose, MOI is multiplicity of infection, PFU is plaque forming units, RGs is reference genes, dpi is days post infection, hpi is hours post infection, N indivs is number of individuals per time point, wk is week.  [15] a Three strains, derived from chicken, egret and duck. b Authors state β-actin was stable between uninfected and infected, but no details given and no other RGs investigated. c Authors state that 18S had the most stable expression over time and between tissues in ducks, but data is not shown and no indication of which RGs were compared. d Five control individuals. e Many results were inferred from graphs because exact results were not listed. In such cases,~is used to indicate fold changes are approximate. f Results not expressed as fold-change. Significant upregulation with one of the two tested viruses only.

Experimental design, materials and methods
To address these methodological issues, in Helin et al. [1] we use a semi-natural infection regime to infect mallards with low pathogenic H1N1 AIV. We then use a set of reference genes (Tables 2 and  3), that we have previously demonstrated to be stable under these experimental settings [5], to normalize RT-qPCR data. A full description of the experimental design, materials and methods is provided in Helin et al. [1].
Datasets describing the fold-change in expression between experimental time-points, and per individual, for each tissue type and gene are provided as Supplementary tables S1-4 and Figs. S1-S4 to this article. Fig. S5 provides a more in-depth analysis of two individuals with extremely high expression, showing that this over-expression was restricted to a specific tissue and a single gene at single time-point. Table 3 Primers used in [1]. F denotes the forward primer and R the reverse primer. Annealing temperature (Ta) expressed in°C and length in base pairs (bp).

Gene Symbol Gene Name
Primers Ta Length

Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at https://doi.org/ 10.1016/j.dib.2018.04.061.