LC–MS/MS Metabolomics Explore Key Pathways Driving HPAI H5N1 Infection in Chickens

The high prevalence of highly pathogenic avian influenza (HPAI) H5N1, a significant pathogen possessing the potential to cause pandemics, combined with a recent expansion in host range, highlight the crucial need to grasp the molecular mechanisms underlying its pathogenesis and host-pathogen interactions.

Metabolomics, the comprehensive study of small-molecule metabolites within biological systems, suggests a method for unraveling these mechanisms to assist in the development of effective control strategies against HPAI H5N1. A joint study conducted by the Indian Council of Agricultural Research (ICAR, Bophal, India), the Yenepoya Research Centre (Mangalore, India), and Tamil Nadu Veterinary and Animal Sciences University (Salem, India) set out to investigate the metabolomic alterations associated with HPAI H5N1 infection. To do so, serum and lung samples were collected by the team from specific pathogen-free (SPF) chickens that were either infected with HPAI H5N1 or mock-infected as controls. Metabolomic profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) under both positive and negative ionization modes. The resulting data were analyzed to identify metabolites that were significantly altered in response to infection. A paper based on this research was published in Frontiers in Cellular and Infection Microbiology (1).

A member of the Orthomyxoviridae family and the genus Influenza virus, influenza virusesare RNA viruses characterized by a segmented, single-stranded, negative-sense genome (2). These viruses are classified into four types: A (avian influenza viruses, IAV), B (infectious bronchitis viruses, IBV), C (influenza C viruses, ICV), and D (influenza D viruses, IDV), with type A especially recognized as a pathogen of significant clinical importance, making it a considerable threat to both the poultry and humans (3). AIVs are categorized based on their effect on chicken, as concluded through the administration of an intravenous pathogenicity index (IVPI) test, into highly pathogenic avian influenza viruses (HPAIV) and low pathogenic avian influenza viruses (LPAIV) (4). Recent incidents of HPAIV strains such as H5N1, H7N9, and H5N8 spreading across a variety of host species has become a serious public health concern (5), with H5N1 particularly notable because of its high pathogenicity, which can lead to significant mortality rates in both chicken and humans (6).

The HPAIV subtype H5N1 is already potentially pandemic in poultry, which can result in severe economic impacts, and can advance to cross species barriers, infecting humans and other mammals, often with fatal outcomes (7). Furthermore, avian influenza viruses, including H5N1, are known to mutate rapidly (7). Therefore, in the opinion of the research team, there is a need for a deeper understanding of the host-pathogen interaction of HPAI H5N1 due to its pervasiveness, capability of human infection, and the potential for mutation. For this purpose, metabolomics offers the ability to come to this understanding as it is a more effective means of comprehending the progression of a disease as opposed to other omics techniques (for example, genomics, transcriptomics, and proteomics), because of its association with phenotype and real-time biological processes (1).

The researchers’ metabolomic analysis revealed substantial changes in both lung and serum samples following HPAI H5N1 infection. Specifically, 31 and 13 altered metabolites were identified in the lung, and 22 and 15 in the serum, under positive and negative ionization modes, respectively. Key metabolites such as sphingosine, psychosine sulfate, and L-serine, which are known to influence viral endocytosis and cell signaling, were significantly altered in infected chickens (1).

The observed changes in sphingolipid and tryptophan metabolism, offer insights into the mechanisms underlying lung and central nervous system (CNS) pathology associated with HPAI H5N1 infection. The identification of specific metabolite alterations may guide future research aimed at mitigating the impact of this highly pathogenic virus (1).

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References

1. Kadamthodi, A. M.; Panwar, A.; Shrungeswara, A. H. et al. Metabolomic Profiling and Identification of Potential Biomarkers of Highly Pathogenic Avian Influenza (H5N1) in Chicken. Front. Cell Infect. Microbiol. 2025, 15, 1540290. DOI: 10.3389/fcimb.2025.1540290
2. Cargnin Faccin, F.; Perez, D. R. Pandemic Preparedness Through Vaccine Development for Avian Influenza Viruses. Hum. Vaccin. Immunother. 2024, 20 (1), 2347019. DOI: 10.1080/21645515.2024.2347019
3. Zhang, Y.; Li, L.; Xin, X. et al. Effects of H9N2 Avian Influenza Virus Infection on Metabolite Content and Gene Expression in Chick DF1 Cells. Poult. Sci. 2024, 103 (10), 104125. DOI: 10.1016/j.psj.2024.104125
4. Liu, Y.; Wei, Y.; Zhou, Z. et al. Overexpression of TRIM16 Reduces the Titer of H5N1 Highly Pathogenic Avian Influenza Virus and Promotes the Expression of Antioxidant Genes through Regulating the SQSTM1-NRF2-KEAP1 Axis. Viruses 2023, 15 (2), 391. DOI: 10.3390/v15020391
5. Sutton, T. C. The Pandemic Threat of Emerging H5 and H7 Avian Influenza Viruses. Viruses 2018, 10 (9), 461. DOI: 10.3390/v10090461
6. Kim, J. H.; Cho, C. H.; Shin, J. H. et al. Highly Sensitive and Label-Free Detection of Influenza H5N1 Viral Proteins Using Affinity Peptide and Porous BSA/MXene Nanocomposite Electrode. Anal. Chim. Acta 2023, 1251, 341018. DOI: 10.1016/j.aca.2023.341018
7. Yamaji, R.; Saad, M. D.; Davis, C. T. et al. Pandemic Potential of Highly Pathogenic Avian Influenza C lade 2.3.4.4 A(H5) Viruses. Rev. Med. Virol. 2020, 30 (3), e2099. DOI: 10.1002/rmv.2099