Influenza A virus

The term “aerosol” is used herein to describe respiratory droplets of all sizes. The term “droplet nuclei” is used to refer to droplets that remain airborne (typically less than 5 μm in diameter).
Each transmission experiment involved eight guinea pigs. On day 0, four of the eight guinea pigs were inoculated intranasally with 10³ PFU of influenza A/Panama/2007/99 virus (150 μl per nostril in phosphate buffered saline [PBS] supplemented with 0.3% bovine serum albumin [BSA]) and housed in a separate room from the remaining animals. At 24 h p.i., each of the eight guinea pigs was placed in a “transmission cage”, a standard rat cage (Ancare R20 series) with an open wire top, which has been modified by replacing one side panel with a wire grid. The transmission cages were then placed into the environmental chamber (Caron model 6030) with two cages per shelf, such that the wire grids opposed each other (Figure 1). In this arrangement, the guinea pigs cannot come into physical contact with each other. Each infected animal was paired on a shelf with a naïve animal. The guinea pigs were housed in this way for 7 d, after which they were removed from the chamber and separated. On day 2 p.i. (day 1 post-exposure) and every second day thereafter up to day 12 p.i., nasal wash samples were collected from anesthetized guinea pigs by instilling 1 ml of PBS-BSA into the nostrils and collecting the wash in a Petri dish. Titers in nasal wash samples were determined by plaque assay of 10-fold serial dilutions on Madin Darby canine kidney cells. Serum samples were collected from each animal prior to infection and on day 17 post-infection, and seroconversion was assessed by hemagglutination inhibition assay.
All transmission experiments reported herein were performed between September 2006 and April 2007.

We performed a systematic search for cohorts from the Gene Expression Omnibus (GEO) (Edgar et al. 2002 (link)) database satisfying the following inclusion criteria: (i) cohorts with bacterial infections or viral infections; (ii) cohorts with hospitalization and clinical information; and (iii) cohorts with whole blood samples. Samples were excluded due to (i) sarcoid and cancer; (ii) unknown pathogens; and (iii) coinfection with bacteria and virus (Supplementary Fig. S1). From the 3203 samples across 16 cohorts, we filtered 2680 samples for subsequent analysis (Supplementary Fig. S1 and Table 1). Patients with Escherichia coli, methicillin-resistant Staphylococcus aureus, tuberculosis, influenza A virus subtype H1N1 etc. were included in the current study to identify bacterial infection and viral infection.
The samples of 14 cohorts in the discovery set were divided into 70% (1876) for training and 30% (804) for testing (Table 1). The training set is applied to extract biomarkers and further train classifiers while the test set is employed to evaluate the performance and determine the hyperparameters of bvnGPS. To verify the generalization ability of bvnGPS, 147 patients in GSE21802 and GSE57065 were used for external validation. To demonstrate the robustness and simplicity of the GPS procedure, no additional preprocessing was performed on the raw expression cohorts.

Vero, Vero E6, Vero.DogSLAMtag, which is stably expressing a CDV receptor, canine SLAM (Seki et al., 2003 (link); Sakai et al., 2013 (link)), MDCK and 293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; catalog number 041-30081, Wako, Osaka, Japan) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (catalog number 15140122, Thermo Fisher Scientific, Waltham, MA). In some cases, an antibiotic for Mycoplasma spp., BIOMYC-3 (catalog number PK-CC03-038-1D, Takara Bio, Shiga, Japan), was added to the culture medium at 100-fold dilution. The working stocks of RNA viruses were prepared as described and were aliquoted and stored at −80°C.
Lymphocytic choriomeningitis virus (LCMV) strain WE (Genbank Accession Numbers LC413283 and LC413284) was propagated in Vero cells at a multiplicity of infection (MOI) of 0.01. The culture supernatants were harvested at 4 days post-infection. The infectious dose was determined using Vero cells with the standard 50% tissue culture infectious dose (TCID₅₀) assay, with visualization of infection on the wells in a 96-well plate by an indirect immunofluorescence assay (IFA), as described previously (Taniguchi et al., 2020 (link)).
Severe fever with thrombocytopenia syndrome virus (SFTSV) strain YG-1 (Genbank Accession Numbers AB817979, AB817987, and AB817995) was propagated in Vero cells at an MOI of 0.01. The culture supernatants were harvested at full cytopathic effect (CPE). The infectious dose was determined with the standard TCID₅₀ assay, with visualization of infection on the wells in a 96-well plate by an IFA, as described previously (Takahashi et al., 2014 (link)).
Influenza A virus (IAV) strain H1N1 A/PR/8/34 (Genbank Accession Numbers LC662537, LC662538, LC662539, LC662540, LC662541, LC662542, LC662543, and LC662544) purchased from ATCC was propagated in MDCK cells with the addition of 1.0 μg/ml trypsin (catalog number 207-19183, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) in DMEM and passaged twice at an MOI of 0.01. The culture supernatants were harvested 3 days post-infection, and the infectious dose was determined using MDCK cells with the standard TCID₅₀ assay in a 96-well plate.
Canine distemper virus (CDV) strain CYN07-dV (Genbank Accession Number AB687720) was propagated in Vero.DogSLAMtag cells at an MOI of 0.01. The cells and culture supernatants were harvested at full CPE and frozen and thawed twice, which is necessary to release the cell-associated virus into the culture supernatant. The samples were centrifuged at 1,000 ×g for 10 min, and the infectious dose was determined with the standard TCID₅₀ assay in a 96-well plate.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain 2019-nCoV/Japan/TY/WK-521/2020 (GISAID ID: EPI_ISL_408667) was propagated in VeroE6 cells stably expressing transmembrane serine protease TMPRSS2 (VeroE6/TMPRSS2) (Matsuyama et al., 2020 (link)) at an MOI of 0.1. The culture supernatants were harvested at full CPE, and the infectious dose was determined using VeroE6/TMPRSS2 cells with the standard TCID₅₀ assay in a 96-well plate.
Pteropine orthoreovirus (PRV) strain Miyazaki-Bali/2007 (Genbank Accession Numbers AB908278.1, AB908279.1, AB908280.1, AB908281.1, AB908282.1, AB908283.1, AB908284.1, AB908285.1, AB908286.1, and AB908287.1) was propagated in 293T cells at an MOI of 0.001. The culture supernatants were harvested at full CPE and titrated using Vero cells with the standard TCID₅₀ assay in a 96-well plate.