# Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

## TL;DR

This study explores how raw milk affects the replication and host response of H5N1 avian influenza in different cell types, revealing strain-specific effects on viral evolution and host gene expression.

## Contribution

The study identifies strain-specific viral replication and host gene modulation effects of H5N1 in the presence of raw milk across diverse cell lines.

## Key findings

- Bovine-derived H5N1 replicates more robustly than mink-derived H5N1 and shows intra-host viral evolution.
- Raw milk modulates host gene expression, dampening antiviral responses and disrupting cellular processes like sialic acid biosynthesis.
- Host gene modulations are cell-type- and strain-specific, suggesting complex virus–host interactions.

## Abstract

The highly pathogenic avian influenza (HPAI) H5N1 virus poses a growing global health threat, particularly following its unprecedented spillover into dairy cattle and subsequent transmission to more than 1000 dairy farms in 18 states. This study investigates the host cell responses to distinct H5N1 strains (bovine- and mink-derived H5N1) in the presence and absence of raw milk across diverse mammalian cell lines (MDCK, MDBK, A549, Vero, MV1). Our findings reveal that the bovine-derived H5N1 strain exhibits more robust replication than the mink-derived H5N1 and demonstrates intra-host viral evolution with emerging amino acid substitutions detectable by deep sequencing. Although raw milk supplementation did not directly enhance viral replication in vitro, it significantly modulated host gene expression, often dampening key antiviral interferon-stimulated gene (ISG) responses and disrupting essential host cellular processes like intracellular trafficking and sialic acid biosynthesis. These host gene modulations are cell-type- and strain-specific, suggesting a complex interplay that may theoretically influence virus–host dynamics, though the biological significance of these in vitro observations requires validation through infectious virus assays and in vivo studies. This hypothesis-generating work provides preliminary insights into H5N1-milk interactions, highlighting the need for further mechanistic investigation to assess potential implications for viral transmission in dairy environments.