| Construction Protocol | Virus Strains: A/Mallard/British Columbia/500/2005 (H5N2) was used for LPAI challenge. A/Vietnam/1203/2004 (H5N1) was used for HPAI challenge. The viruses were grown in the allantoic cavities of 10-day-old embryonated chicken eggs for 48 hours at 35 degrees C. Allantoic fluid containing virus was harvested and stored at -80 degrees C until use. Virus yield was determined as 50% egg infectious dose (EID50) per millilitre of virus stock by the method of Reed and Muench. Experimental Animals: Specific pathogen–free white leghorn chickens were purchased from Charles River Laboratories (North Franklin, CT). All experiments involving animals were approved by the Animal Care and Use Committee of St. Jude Children's Research Hospital and performed in compliance with relevant policies of the National Institutes of Health and the Animal Welfare Act. All animal challenge experiments were performed in an animal biosafety level 2 containment facilities for the LPAI challenges and in biosafety level 3 enhanced containment laboratories for the HPAI challenges. Viral Challenge: Chickens, except the mock infection control group, were challenged with 106 EID50 intranasally, intratracheally, and intraocularly of LPAI A/Mallard/British Columbia/500/2005 (H5N2) in phosphate buffered saline (PBS). Chickens, except the mock infection control group, were challenged with 101.5 EID50 intranasally, intratracheally, and intraocularly of HPAI A/Vietnam/1203/2004 (H5N1) in PBS. During LPAI and HPAI infections in chickens and ducks, mock infection control groups were also inoculated. Mock infection control birds received an equivalent volume and route of administration with PBS. Animals were monitored daily for clinical signs. Tissue Sampling: Lung and ileum samples were collected from all birds on days 1 and 3 dpi. Tissue homogenates were inoculated into 10-day-old embryonated chicken eggs in triplicate to screen for positive samples. Positive samples were then titrated in eggs and expressed as log10EID50/mL. The lower limit of detection was 0.75 log10EID50/mL. For each ileum and lung tissue, the tissue sample was homogenized in Trizol (Invitrogen Life Technologies), 0.1g tissue/1.0ml Trizol with handheld homogenizer Power Gen125 (Thermo Fisher Scientific). After homogenates were prepared, the samples were centrifuged to separate the liquid phases of Trizol homogenates. 0.5ml of the aqueous phase was passed through a QIagen RNeasy (Qiagen, Valencia, CA) spin column according to manufacturer's instructions for RNA extraction. Samples were frozen and stored at -80 degrees C until all samples were collected for analysis. Samples were prepared for mRNA sequencing using 5ug of total RNA starting material following the Illumina mRNA sequencing 8 sample preparation kit protocol. |
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