Protocol for virome characterization in low-volume respiratory samples from broiler chickens

The poultry industry is a major global source of animal protein but remains vulnerable to immunosuppressive viral infections that compromise bird health and productivity. This study evaluated five viral purification methods for metagenomic analysis of respiratory samples from broiler chickens in Santa Catarina, Brazil. Tracheal swabs from ten flocks (one per farm) were pooled, and 50 µL of a herpes simplex virus type 2 (HSV-2) and murine norovirus (MNV-1) mix was added as an internal positive control. The sample was centrifuged (2000 × g for 30 min), filtered (0.45 μm), and subjected to five purification methods. The filtrate was subjected to five different purification methods. Method 1 (M1) was based on nucleic acid direct genomic extraction of the supernatant. Method 2 (M2): a pre-treatment with DNase was used, followed by genomic extraction. Method 3 (M3) was performed using ultracentrifugation at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. In Method 4 (M4), the sample was submitted to ultracentrifugation on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. Finally, in Method 5 (M5), the sample was ultracentrifuged on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, and the pellet was treated with DNase followed by genomic extraction. All genomic extractions were performed using the RNeasy Mini kit. Samples were reverse transcribed into cDNA and sequenced by the MiSeq Sequencing System. The efficiency of M1-5 was evaluated based on the yield of viral genetic material. All methodologies employed demonstrated varying rates of genome recovery from viruses identified in poultry production. Notable viruses included avian gyrovirus 2 (AGV-2), avian leukosis virus (ALV), and the avian endogenous retrovirus EAV-HP found within chicken genomes. However, M5 showed the best performance, recovering 9.32 % of viral sequences, 44 % of HSV-2, as internal viral control, 32 % of EAV-HP, 8 % of ALV, and 7 % of AGV-2. In conclusion, this study successfully evaluated and compared five distinct viral purification methods, contributing significantly to the characterization of avian viromes and enhancing comprehension of viral ecology.