Experimental adaptation of an influenza H5 haemagglutinin (HA) confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets methods
Aim. Evidence-backed execution summary for Experimental adaptation of an influenza H5 haemagglutinin (HA) confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets methods from Experimental adaptation of an influenza H5 haemagglutinin (HA) confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- Madin-Darby canine kidney (MDCK) cells were maintained in Eagle's minimal essential medium containing 5% newborn calf serum in 5% CO 2 at 37°C.
Experimental infection of ferrets
reagent used in the protocol.
- Use
- We used 6- to 10-month-old female ferrets (Triple F Farms) that were serologically negative by HI assay for currently circulating human influenza viruses. Six ferrets per group were anesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg per kg of body weight, respectively) and inoculated intra...
Experimental infection of ferrets
reagent used in the protocol.
- Use
- Excised tissue samples of nasal turbinates, trachea, lungs, brain, liver, spleen, kidney, and colon from euthanized ferrets were preserved in 10% phosphate-buffered formalin. Tissues were then trimmed and processed for paraffin embedding and cut into 5-µm thick sections. One section from each tissue sample was...
Serologic tests
reagent used in the protocol.
- Use
- Serum samples were collected between days 14 and 20 post-infection, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by use of an HI assay with 0.5% turkey red blood cells (TRBCs). Viruses bearing homologous HA were used as antigens for the HI test.
NA inhibition assay
reagent used in the protocol.
- Use
- To assess the sensitivity of viruses to the NA inhibitor oseltamivir, NA inhibition assays were performed as described previously.
Solid-phase binding assay
reagent used in the protocol.
- Use
- Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays w...
Virus binding to human airway tissues
reagent used in the protocol.
- Use
- Paraffin-embedded normal human trachea (US Biological) and lung (BioChain) tissue sections were deparaffinized and rehydrated. Sections were then blocked by using 4% BSA in PBS and covered with virus suspensions (64 HA units in PBS) at 4°C overnight. After being washed four times in ice-cold PBS, the sections w...
Experimental infection of ferrets
Excised tissue samples of nasal turbinates, trachea, lungs, brain, liver, spleen, kidney, and colon from euthanized ferrets were preserved in 10% phosphate-buffered formalin. Tissues were then trimmed and processed for paraffin embedding and cut into 5-µm thick sections. One section from each tissue sample was...
- Use
- Excised tissue samples of nasal turbinates, trachea, lungs, brain, liver, spleen, kidney, and colon from euthanized ferrets were preserved in 10% phosphate-buffered formalin. Tissues were then trimmed and processed for paraffin embedding and cut into 5-µm thick sections. One section from each tissue sample was...
Solid-phase binding assay
Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays w...
- Use
- Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays w...
Virus binding to human airway tissues
Paraffin-embedded normal human trachea (US Biological) and lung (BioChain) tissue sections were deparaffinized and rehydrated. Sections were then blocked by using 4% BSA in PBS and covered with virus suspensions (64 HA units in PBS) at 4°C overnight. After being washed four times in ice-cold PBS, the sections w...
- Use
- Paraffin-embedded normal human trachea (US Biological) and lung (BioChain) tissue sections were deparaffinized and rehydrated. Sections were then blocked by using 4% BSA in PBS and covered with virus suspensions (64 HA units in PBS) at 4°C overnight. After being washed four times in ice-cold PBS, the sections w...
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Experimental infection of ferrets
We used 6- to 10-month-old female ferrets (Triple F Farms) that were serologically negative by HI assay for currently circulating human influenza viruses. Six ferrets per group were anesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg per kg of body weight, respectively) and inoculated intranasally with 10 6 PFU (500 µl) of viruses. On days 3 and 6 post-infection, 3 ferrets per group were euthanized for virologic and pathologic examinations. The virus titres in various organs were determined by plaque assays in MDCK cells.
Serologic tests
Serum samples were collected between days 14 and 20 post-infection, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by use of an HI assay with 0.5% turkey red blood cells (TRBCs). Viruses bearing homologous HA were used as antigens for the HI test.
Solid-phase binding assay
Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays with 0.5 % (vol/vol) TRBC. The direct receptor-binding capacity of viruses was examined by use of a solid-phase binding assay as previously described. Microtitre plates (Nunc) were incubated with the sodium salts of sialylglycopolymers [poly-L-glutamic acid backbones containing N -acetylneuraminic acid linked to galactose through either an α2,3 (Neu5Acα2,3Galβ1,4GlcNAcβ1-pAP) or an α2,6 (Neu5Acα2,6Galβ1,4GlcNAcβ1-pAP) bond] in PBS at 4°C overnight. After the glycopolymer solution was removed, the plates were blocked with 0.15 ml...
Measurement outputs
What raw and processed outputs should exist?
All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.). The statistical significance of differences between pdm09, H5HA/pdm09, and H5HA-mutant/pdm09 viruse...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.).
from paperScoring or quantification
Quantify the primary readouts for this experiment: All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.). The statistical significance of differences between pdm09, H5HA/pdm09, and H5HA-mutant/pdm09 viruse....
from paperStatistical comparison
All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.). The statistical significance of differences between pdm09, H5HA/pdm09, and H5HA-mutant/pdm09 viruse...
from paperReporting output
Report representative outputs alongside summary comparisons for All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.). The statistical significance of differences between pdm09, H5HA/pdm09, and H5HA-mutant/pdm09 viruse....
inferred from protocolStructured statistical methods
All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.). The statistical significance of differences between pdm09, H5HA/pdm09, and H5HA-mutant/pdm09 viruse...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
We used 6- to 10-month-old female ferrets (Triple F Farms) that were serologically negative by HI assay for currently circulating human influenza viruses. Six ferrets per group were anesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg per kg of body weight, respectively) and inoculated intranasally with 10 6 PFU (500 µl) of viruses. On days 3 and 6 post-infection, 3 ferrets per group were euthanized for virologic and pathologic examinations. The virus titres in various organs were determined by plaque assays in MDCK cells.
Serum samples were collected between days 14 and 20 post-infection, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by use of an HI assay with 0.5% turkey red blood cells (TRBCs). Viruses bearing homologous HA were used as antigens for the HI test.
Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays with 0.5 % (vol/vol) TRBC. The direct receptor-binding capacity of viruses was examined by use of a solid-phase binding assay as previously described. Microtitre plates (Nunc) were incubated with the sodium salts of sialylglycopolymers [poly-L-glutamic acid backbones containing N -acetylneuraminic acid linked to galactose through either an α2,3 (Neu5Acα2,3Galβ1,4GlcNAcβ1-pAP) or an α2,6 (Neu5Acα2,6Galβ1,4GlcNAcβ1-pAP) bond] in PBS at 4°C overnight. After the glycopolymer solution was removed, the plates were blocked with 0.15 ml of PBS containing 4% bovine serum albumin (BSA) at room temperature for 1 h. Following four successive washes with ice-cold PBS, the plates were incubated in a solution containing influenza virus (8 to 32 HA units in PBS) at 4°C overnight. After washing as described above, the plates were incub...
Machine-readable layer
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