Gene function & annotation

An in silico-in vitro workflow to validate useful genomic variants against intestinal infectious diseases in pigs

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Abstract

Inbreeding and loss of genetic diversity limit the ability of populations to adapt to recurring and new emerging pathogens. Breeding schemes enabling the maintenance of genetic diversity at key immune host response loci could provide pig breeders with an important and durable layer of protection. In silico predictions identify and predict the effect of candidate functional variants, but the further use of this information for breeding programmes requires suited validations. Here, we aim to predict and validate genomic variants in pan-virus host restriction factors that orchestrate the early stages of viral infection and coinfection, using a pipeline that integrates bioinformatic prediction tools with an appropriate in vitro model. By mining the abundant information available for Coronaviruses, we identified 150 selected porcine loci modulating the immune response to the main recurrent porcine enteric coronaviruses. The Transmissible Gastroenteritis virus (TGEV) is known to cause strong losses to pig productions, either directly or by predisposing to secondary infections. As the ANPEP gene encodes the primary TGEV receptor, we used it as a model to assess the validity of our pipeline. We mined ANPEP variants by means of open-source databases, then used VEP and Pangolin programs to predict their functional impact. While VEP provides broad variant annotations and consequences across transcripts and proteins, Pangolin specifically focuses on predicting the effects of variants on RNA splicing using deep learning models. Variants predicted to significantly alter protein domains involved in the entry of TGEV (Exon 15 to 17 of the canonical ANPEP transcript), were selected for further experimental validation. As in vitro system we used organoids derived from the jejunum of 4 pre-weaned piglets by established in-house protocols. Thirty-two variants located in the region spanning exons 15 to 17, including the flanking 5′ and 3′ intronic regions, were retrieved from the European Variation Archive (release 8). Experimentally, we first optimized the protocols for TGEV infection of organoids. Immunofluorescence analysis revealed infection rates ranging from 3.1% to 18.5%, showing individual variation. To validate the specificity of infection, we generated ANPEP knock-out organoids, which showed no detectable signal following TGEV exposure. These organoids were used as negative controls to calibrate the in vitro infection system. Results showed that TGEV infection on the heterogeneous epithelial cell population composing jejunum organoids represents a promising in vitro system to qualify and quantify the effect of variants at immune-associated gene loci in pig.

Keywords: 2026

How to Cite:

Egidy, G., Charles, M., Carbonne, C., Coville, J., Boulling, A., Delmas, B., Demars, J., Ferchaud, S., Grivault, D., Lejal, N., Mercat, M., Meunier, N., Nevers, Q., Restoux, G., Servin, B., Taussat, S., Tribout, T. & Giuffra, E., (2026) “An in silico-in vitro workflow to validate useful genomic variants against intestinal infectious diseases in pigs”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2295349. doi: https://doi.org/10.31274/wcgalp.24338

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