Analyzing the role of the retinoic acid-inducible gene I (RIG-I) in response to avian influenza virus infection using transgenic chicken models
Final Report Abstract
The world is in the middle of a global pandemic of avian flu. Since the summer of 2023, almost 70 countries over five continents reported the spread of H5N1, which caused the death of more than 130 million domestic birds. The World Health Organization (WHO) is warning of the potential risk to humans due to rapid mutations and the increasing number of H5N1 avian influenza detections among mammals (WHO). The zoonotic potential of the virus and its ability to mutate quickly call for an urgent understanding of host-pathogen interactions. Chickens are the natural avian influenza host and the most consumed livestock worldwide. Hence, they offer a unique opportunity to study the interaction of avian influenza with the host. Unlike most vertebrates, the chicken has lost the immune sensor RIG-I over evolution, while this gene was well-reserved in wild ducks. The reason behind the disappearance of RIG-I in most Galliformes remained enigmatic till we conducted this project. RIG-I is a cytosolic RNA sensor that recognizes and binds to the 5’ triphosphate end (5’-ppp). It forms a first line of antiviral defense as a pathogen recognition receptor (PRR) against RNA viruses. Upon activation, the RIG-I interacts with the mitochondrial antiviral signaling proteins (MAVS), inducing a pro-inflammatory antiviral response characterized by the upregulation of type I and type III interferons (IFNs) followed by the expression of IFN stimulated genes (ISGs). The activity of RIG-I is believed to be controlled by post-translational modification of tripartite motifcontaining protein 25 (TRIM25) and RING finger protein 135 (RNF135, also known as Riplet or REUL). The latter was found to modify RIG-I by lysine 63-linked polyubiquitination of the C-terminal region of the caspase activation and recruitment domain (CARD), leading to a stronger RIG-I signal transduction. In this work, we had two main goals: 1) to reinstate the RIG-I and its ubiquitination factor RNF135 in the chicken genome in vivo and to examine its effect on different physiological parameters and 2) how it influences the susceptibility towards avian influenza virus. To achieve the first aim, we used primordial germ cells (PGCs) to generate genetically modified chickens expressing duck RIG-I, duck RNF135 or both genes. Both genes were expressed under their respective duck promoter. While the activity of the duck RIG-I promoter was previously investigated (2), we identified the duck RNF135 promoter activity for the first time. We mated heterozygous birds expressing either RIG-I or RNF135 to obtain birds that co-expressed both genes. In the absence of infection, we observed an alternative phenotype of the adaptive immune system of RIG-I-expressing chickens, particularly regarding the T cell population. In contrast, the co-expression of RNF135 with RIG-I contributed to a balanced adaptive immune phenotype that appeared to be similar to WT birds. The generated birds were healthy and developed normally compared to their WT siblings. For the second aim, we wanted to investigate the susceptibility of the generated birds to the avian influenza virus. We conducted infection experiments with AIVs in vitro, in ovo, and in vivo. The outcome was surprising since the infection led to severe clinical disease associated with a strong inflammatory response, high IFN-γ expression, and elevated viral replication in RIG-I-expressing chickens compared to other challenged groups. In contrast, infected RIG-I-RNF135-expressing chickens presented with inflammation limited viral replication, and increased IFN-α expression levels. The obtained data reveal the immunological functions of RIG-I in chickens and the benefit of learning from less susceptible species to influenza infection to improve the immune system’s resilience toward infection.
Publications
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Co-expression of the duck ring finger protein (RNF135) and retinoic acid-inducible gene I (RIG-I) in chicken DF-1 cells leads to high protection against influenza A viruses. European congress of virology, Rotterdam, Netherlands 28.04-01.05.2019
Sid H., Heymelot L. & Schusser B.
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Von der Ente lernen: neue Möglichkeiten für das Immunsystem des Huhns. Geflügelfachgespräch, Hannover, Germany, 2022
Sid H., von Heyl T., Alhussien M., Schleibinger S., Vikkula H. & Schusser B.
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Generation of RIG-I expressing chickens and their susceptibility to influenza A virus. XII World Veterinary Poultry Association Verona, Italy 4-8 September 2023
Sid H., von Heyl T., Guabiraba R., Sives S., Vervelde L., Trapp S. & Schusser B.
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Transgenic chickens re-expressing evolutionarily lost immune sensors: Spotlight on the evolution of the avian immune system. Transgenic Animal Research Conference XIV UC Davis, California, USA 13-17 August 2023
Sid H., von Heyl T., Guabiraba R., Sives S., Vervelde L., Trapp S. & Schusser B.
