NIH grant: Host-specific profiles of Defective Viral Genomes of influenza virus (1R21AI199873-01)

retained text

NIH grant 1R21AI199873-01: Host-specific profiles of Defective Viral Genomes of influenza virus
Institute: {'code': 'AI', 'abbreviation': 'NIAID', 'name': 'National Institute of Allergy and Infectious Diseases', 'admin_org_id': '12500003', 'admin_funding_url': ''}. PI: Samuel L Diaz. Award: $270443.
Start: 2026-06-23T00:00:00.
PROJECT SUMMARY
RNA viruses are exceptionally diverse and rapidly evolving. Their RNA-dependent RNA polymerases are prone
to mutation, lack proof-reading and frequently undergo recombination. Non-homologous RNA recombination
gives rise to Defective-Viral Genomes (DVGs); versions of the parental genome that have been truncated,
internally deleted or otherwise rearranged. While not encoding for functional viruses, DVGs can be amplified and
co-passaged with the wild-type ‘helper’ virus that provides the machinery for replication, encapsidation and
transmission. DVGs spontaneously emerge during viral replication and can subsequently act as ‘parasites’ or
‘cheats’ of the parental virus by competing for cellular resources and viral cofactors. DVGs can change the
fitness, outcomes and vaccine effectiveness of respiratory pathogens such as influenza A virus (IAV), respiratory
syncytial virus (RSV) and SARS-CoV-2. Recent studies have characterized the ability of DVGs to modulate
immune responses. In particular, IAV generates DVGs primarily from PB2, PB1 and PA segments that are known
to activate RIG-I pathways and simulate interferon-B expression. This results in cytokine stimulation and cell-
death and may explain the variable pathogenicity of some influenza strains. Specific mutations in the polymerase
of H1N1 (PA:D529N) have been identified that correlate with reduced DVG formation, a lowered induction of
innate immunity, and increased pathogenesis in mouse models. In human cohorts, patients with highly severe
or fatal outcomes exhibited a lower ‘dose’ of DVGs relative to mild cases. We recently performed a secondary
analysis of publicly available NGS data of 2421 samples of Highly pathogenic avian influenza (HPAI) (H5N1
clade 2.3.4.4b) collected from poultry, non-poultry and cattle by the USDA (Nguyen et al, 2024). We found a
striking abundance of DVGs in PB2, PB1 and PA segments across the entire cohort. Importantly, we also found
evidence for species-specific DVGs, raising the tantalising prospect that DVGs might be useful as a biomarker
reporting on the biological origins of a virus sample, in the absence of any other information.
We will validate and formalize these findings with a series of bioinformatic and computational routes to identify
a molecular DVG-based signature of the host of origin. We next develop a ClickSeq-based assay, that we will
term ‘FluDVG-ClickSeq’, for targeted influenza viral NGS that can accurately report on the abundance and
identity of DVGs in clinical specimens using simple, automatable bioinformatics. Together, we will determine
whether the DVGs can be used as biomarker to identify the host of origin in complex samples, suchas
environmental collections where a host or infected host is not present or identifiable. In addition to providing a
valuable tool for epidemiology and surveillance efforts, this will provide support for the generalization concept
that DVGs might form in a host-dependent manner.

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