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Protocol for virome characterization in low-volume respiratory samples from broiler chickens
Giulia Von Tönnemann Pilati1, Henrique Borges da Silva Grisard1, Rafael Cadamuro Dorighello1
1Laboratory of Applied Virology, Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianópolis 88040-900, Brazil.
Journal of Virological Methods
|August 1, 2025
Summary
This study compared five viral purification methods for poultry respiratory samples. Method 5, involving ultracentrifugation and DNase treatment, yielded the highest recovery of viral genetic material, improving avian virome characterization.
Area of Science:
- Veterinary Virology
- Metagenomics
- Poultry Health
Background:
- Poultry industry faces economic losses due to viral infections.
- Effective viral detection is crucial for maintaining flock health and productivity.
- Metagenomic analysis offers a comprehensive approach to studying complex viral communities.
Purpose of the Study:
- To evaluate and compare five distinct viral purification methods for metagenomic analysis of broiler chicken respiratory samples.
- To determine the most efficient method for viral genome recovery in poultry.
- To enhance the characterization of the avian virome.
Main Methods:
- Five viral purification methods (M1-M5) were tested on pooled tracheal swabs from broiler chickens.
- Methods included direct extraction, DNase treatment, ultracentrifugation, and sucrose cushion ultracentrifugation.
- Herpes simplex virus type 2 (HSV-2) and murine norovirus (MNV-1) were used as internal controls.
- Viral genetic material yield was assessed using the RNeasy Mini kit, cDNA synthesis, and MiSeq sequencing.
Main Results:
- All tested methods recovered viral genetic material with varying efficiencies.
- Method 5 (ultracentrifugation on a sucrose cushion with DNase treatment) demonstrated superior performance.
- Method 5 achieved the highest overall viral sequence recovery (9.32%), including significant recovery of internal controls (HSV-2: 44%) and relevant poultry viruses (EAV-HP: 32%, ALV: 8%, AGV-2: 7%).
Conclusions:
- Method 5 is the most effective viral purification strategy for metagenomic analysis of poultry respiratory samples.
- Optimized viral purification enhances the accurate identification and characterization of avian viruses.
- This research contributes to a better understanding of poultry viral ecology and disease prevention.
