VirusesSegment sequences of the A/mulard duck/France/171201g/2017 (H5N8) HPAIV field isolate were used to produce a reversed genetic virus (H5N8/HP) (PUBMED accession number: MK859904 to MK859911). Based on these sequences information, a LPAIV was engineered using a site-directed mutagenesis (H5N8/LP) (PUBMED accession number: MK859926 to MK859933). Briefly, a 9-nucleotide deletion was performed on the HA cleavage site flanked with two single nucleotide polymorphisms37. Both viruses were propagated on 9–11 day-old specific-pathogen-free (SPF) embryonated chicken eggs (INRAE, PFIE, Nouzilly, France) by allantoic sac inoculation.Both viruses were genetically identical, except for the sequence encoding the HA cleavage site, allowing us to compare two pathotypes of the same strain.These viruses were produced and used only in biosafety level 3 laboratories at the National Veterinary School of Toulouse (ENVT) France.Viral infection of chicken cell linesDF-1 cell line of chicken embryo fibroblast were provided by the Friedrich Loeffler Institut. DF-1 were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) high glucose with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin–streptomycin) at 37 °C with 5% CO2.24H prior to infection, 9T-75 flasks were set with 7.106 cells/flask. On the day of infection, cell confluency and homogeneity between flasks were assessed. Flasks were randomly divided in three groups of three for the H5N8/HP, H5N8/LP, and the control group. Infection media was prepared using Opti-MEM supplemented with 0.5 mg/mL TPCK-treated trypsin. Infection media was used to prepare the inoculum and to mock viral infection for the control group. Before inoculation, flasks were washed once with phosphate-buffered saline (PBS) and then infected at a multiplicity of infection (MOI) of 10−5. After 1H, all inoculum were removed, and 25.5 mL of fresh infection media was added to each flask.The experiment was repeated in an independent manner five times. The first four were used as training experiments, and the last was used as a test experiment. The test experiment with new cells was performed two months after the training experiment.Cell supernatant collection5.5 mL of cell supernatant was collected at 1H, 6H, 10H, 24H, 48H and 72H. 5 mL were placed in 20 mL glasses vial (ref 180420, BGB Analytik AG, Rohrmattstrasse, Switzerland) with silicone/PTFE septa (Ref 180301, BGB Analytik AG, Rohrmattstrasse, Switerland) and 0.5 mL in 1.5 mL collection tubes (Eppendorf, Hamburg, Germany).Glasses vial were immerged 10 min at 60 °C in a water bath for viral decontamination before being stored at − 20 °C before being used for VOC’s analysis. Collection tubes were stored at − 80 °C before being used for vRNA detection and quantification.RT-qPCR analysisTotal RNA from all collected samples was extracted using the magnetic bead-based kit ID Gene Mag Fast Extraction (Innovative Diagnostics, Grabel, France) associated with the Ideal 96 automated extraction robot (Innovative Diagnostics, Grabel, France) following manufacturer’s instructions. Detection and quantification of viral RNA (vRNA) were performed in a 10µL final volume using the Itaq SYBR green one-step RT-qPCR kit (Bio-Rad, Hercules CA, USA) following manufacturer instructions with the 5′-GACCTCTGTTACCCAGGGAGCCT-3′ and 5′-GGACAAGCTGCGCTTACCCCT-3′ forward and reverse primers, respectively, specific to both H5 hemagglutinin37. The absence of viral contamination was assessed by performing Itaq SYBR green one-step RT-qPCR (Bio-Rad, Hercules CA, USA) using HPAIV and LPAIV specific primers37.vRNA statistical analysisWelsh-adapted t-test was performed for vRNA data analysis.SIFT-MS analysisSample preparationCell supernatant collected at 1H, 10H, 24H 48H, and 72H were selected for SIFT-MS analysis. Samples from all five experiments were injected in separate days. Each experiment samples were injected within 48H, or 72H. The injection order was randomly selected for each experiment.12H before the start of the injection, samples were stored at 6 °C in a refrigerated room. 1H before their injection, samples were placed 30 min at room temperature to allow the VOCs to equilibrate in the vial headspace. Then, they were heated at 37 °C for 30 min in a dry block heater (Ohaus, Parsippany, USA). SIFT-MS injection was made while samples were still heated at 37 °C.SIFT-MS analysisVOCs measurements were performed using a Selected Ion Flow Tube Mass Spectrometer (SIFT-MS) voice 200 ultra (SYFT Technologies, Christchurch, New Zealand) equipped with a dual polarity source of positive and negative precursor ions (H3O+, NO+, O2+, O−, OH−, O2−, NO2−, and NO3−) set to full scan mode (from 15 to 250 m/z). Each full scan includes four successive full mass scans for a duration of 18 min. For each full mass scan, precursor ions were successively selected by a first quadrupole mass filter and injected to the flow tube with Nitrogen as carrier gas (Alphagaz, Air liquid, 99.9999%, Paris, France). Nitrogen flow rate was set at 2.0 Torr.L/s. Air samples were introduced in the instrument with a flow rate of 0.3 Torr.L/s. Dwell time limit was set to 5 ms. Precursor ions and air samples analytes reaction happened in a 199 °C flow tube kept at 0.006 kPa. The reaction generated product ions with specific mass-to-charge ratios (m/z), which were detected by a second quadrupole mass spectrometer. SIFT-MS was calibrated daily before sample analysis with a standard gas (Air liquid America, Specialty Gases LLC, Plumsteadville PA, USA). The calibration is validated by the instrument if the standard gas (i.e. 1,2,3,4 tetrafluoro benzene, benzene, ethylene, isobutane, octafluorotoluene, p xylene, perfluorobenzen, and toluene) concentration are detected at 2 ppm. Data acquisition and analysis were performed by the LabSyft 1.6.2 software (Syft Technologies, Christchurch, New Zealand).Headspace from the 20 mL glasses vials containing cell supernatants were injected in the SIFT-MS through a customized injection line connected to a 5 mL sterile syringe with a 20G needle. Each sample was also connected to a Nitrogen filled Tedlar bag (ref 22050, Restek, Centre County PA, USA). Three Tedlar bags were used for H5N8/HP, H5N8/LP, and control cells group samples, respectively. Even though the Tedlar bags were not changed for the training experiment samples, the absence of contamination was assessed before each experiment. New ones were used for the test experiment samples.Each day, after the instrument calibration, three blanks were performed, one for each sample group, using the appropriate Tedlar bags and empty 20 mL glasses vials.VOCs analysisGeneral data pre-processingBefore complete statistical analysis, all samples from the five experiments were pre-processed altogether using R Statistical Software version 4.1.138. As full scan mode was set to four full mass scans, each sample presented four signal intensities for each product ion. To insure correct analysis, the first of the four full scan was removed as significantly different form the others. Then, the mean of the three last mass scans was calculated for each sample and each production. Therefore, we obtained only one signal intensity value for each product ions per sample. Secondly, background noise was removed by subtracting each corresponding blank from the samples. Negative obtained values were set to zero.This resulted in an XY matrix with X being the samples (n = 225) and Y the product ions defined based on their precursor ions and m/z value (n = 1888). On these data, 28 ions were removed due to a significant clustering effect (Supp data Table 1).Timepoint specific analysisFive subset matrices were created based on sampling time (1H, 10H, 24H, 48H, or 72H) for discrimination and prediction analysis. For each subset matrix, all variables with an intensity value of 0 for all samples in at least one experiment were removed. Then, we used the Combat function39 from the sva packages40 to remove batch effect from the five experiments.Finally, for all five subsets matrix, data were divided into a training data set and test data sets. The training data set regrouped samples from the first four experiments (n = 36), while the test data tested the samples from the fifth experiment (n = 9).sPLS-DA analysisDespite data pre-processing steps, the number of variables is largely more than the number of samples, which is unappreciated for standard analysis. Therefore, we decided to apply dimension reduction analytical methods. Additionally, as the aim of the study was to investigate sample discrimination based on categorical group and assess prediction samples based on selected variables, we decided to apply sparse partial least square discriminant analysis (sPLS-DA) using the user-friendly MixOmics package34,36. sPLS-DA was first used to select the most discriminant variables for group discrimination, and then, selected variables were used to predict the test samples.The sPLS-DA parameters were tuned for each time point analysis. However, as all timepoint group samples are homogeneous in terms of number per group and number of groups, cross-validation parameters were set identically between all timepoint analyses. Folds were set to three, validation method to ‘Mfold’, distance to ‘max.dist’, nrepeat to 50.
