Animal experiments were performed with the approval and supervision of the Experimental Animal Ethics Committee of Jinan University (Guangdong, China). SPF-grade normal C57BL/6 wild-type mice were purchased from the Experimental Animal Center of Guangdong Province (production license SCXK [Guangdong] 2019-0056). Specific-pathogen-free (SPF), 6–8-week-old female C57BL/6J wild type (WT) mice, weighing 20 $\pm$ 2 g, and B6.129S1-Tlr7tm1Flv$>$/J (Tlr7-/-) mice were purchased from China Guangdong Medical Experimental Animal Center and The Jackson Laboratory (Sacramento, CA, USA), respectively. All experimental mice were raised in an animal biosafety level 2 (ABSL-2) laboratory with a temperature-controlled animal feeding facility (temperature: 20 $\pm$ 2 °C; humidity: 50%), a 12-hour light cycle, and an independent ventilation cage (IVC). The endpoint of animal health is that the animal is moribund, or does not respond after being given a gentle stimulus; Dyspnea: typical symptoms are salivation and/or cyanosis of the nose and mouth; diarrhea or urinary incontinence; A clear sign of serious illness, at which point the animal is euthanized to avoid further unnecessary suffering. After placing the laboratory animals to be euthanized in the box, carbon dioxide is released to suffocate the experimental animals.

Thirty WT and 30 Tlr7-/- mice were randomly divided into five groups: normal control (untreated), virus control (Virus), antibiotic treatment + virus (Model), model + probiotic recovery (Probiotics), and model + fecal microbiota transplantation (FMT). Model, Probiotics, and FMT groups were treated with antibiotics mixed in drinking water for 25 days. All mice were fed sterilized rat food. On day 26, feces from each group of mice were collected from their sterile containers; mice were then anesthetized using a 1% pentobarbital sodium intraperitoneal injection. The untreated group was treated with a 50 $\mu$L sterile saline nasal drip, while the other groups were infected with 50 $\mu$L of an influenza virus FM1 suspension. On day 27, the Probiotics group was treated with probiotics via intragastric administration for five days, while mice in the FMT group were transplanted with untreated mice feces for five consecutive days; other groups were fed normal saline for five days. On day 32, sacrifice mice by euthanasia. Each group of lungs, spleen, cecum, and feces were collected for analysis (Fig. 1a).

Fig. 1.

The changes of body weight and lung index, pathological analysis and virus replication were observed. (a) is experimental flow graph. (b) and (c) represent changes to body weight and lung index, respectively, in each group of WT mice (n = 6). (d) and (e) represent changes to body weight and lung index, respectively, in Tlr7${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice of each group (n = 6). (f) represent pathological sections of lung and cecum tissues of WT mice and Tlr7${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice. Bars = 200 $\mu$m. The black arrow points to the mucosa of the cecum. (g) and (i) represent the titer of FM1 virus in the lung tissue of WT and Tlr7-/- mice on day 6 after infection (n = 6). (h) and (j) represent FM1 virus mRNA expression in the lung tissue of WT and Tlr7-/- mice on day 6 after infection (n = 6). *p 0.05, **p 0.01, ***p 0.001, NS p $>$ 0.05.

The antibiotic drinking water mix was prepared by adding ampicillin (1 mg/mL, Lot: 7918010110), neomycin (1mg/mL, Lot: 9311010150), metronidazole (1mg/mL, Lot: c10473109), gentamicin (0.5 mg/mL, Lot: H04J10Z89946), and vancomycin (0.5 mg/mL, Lot: H26M10Z84068) (all from Guangzhou Dingguo Biotechnology Co., Ltd., Guangzhou, China) to sterilized pure drinking water; this was replaced every three days.

Bifidobacterium and Lactobacillus triple live bacteria tablets (Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., Inner Mongolia,China , Chinese medicine S19980004) were used strictly according to the manufacturer’s instructions; the daily dosage for each mouse was 0.78 g/kg. Probiotics were dissolved in sterilized saline and administered intragastrically at 0.2 mL/d [12, 13].

Twenty-six days after antibiotic treatment, before being infected with virus, anus of each mouse was wiped with cotton swabs to stimulate defecation on a sterile container collecting feces. On day 32, following euthanasia, fecal samples were collected from the rectum of mice by opening the abdominal cavity using sterilized equipment in a biosafety cabinet. Collected feces were placed into sterile EP tubes and frozen at –80 °C. All procedures were completed within 30 min. The samples were sent to Guangdong Magigene technology Co., Ltd (Guangzhou, China) for testing.

The 16S rRNA gene was sequenced by Guangdong Magigene Biotechnology Co., Ltd (HiSeq2500/Miseq, PE250/PE300, Guangdong, China). Genomic DNA was extracted using the ALFA-SEQ Advanced Stool DNA Kit; the concentration and purity of DNA were detected using NanoDrop gel One. The hypervariable region of 16S rDNA V4–V5 was amplified using polymerase chain reaction (PCR), with barcode-tagged primers and TaKaRa Premix Taq® Version 2.0 (RR901A, TaKaRa, Guangzhou, China). Mixed PCR products were recovered using an E.Z.N.A.® Gel Extraction Kit (Omega, Nocross, Georgia, USA); target DNA fragments were recovered via elution with TE buffer. The Illumina Nova 6000 platform was used for PE250 sequencing of the expanded sublibrary. Head and tail primers were simultaneously removed using Cutadapt software, thus obtaining quality-controlled, paired-end clean reads. Fastp was used to tailor raw tag data with sliding window quality (– W4–M20), thus obtaining an effective splicing segment (clean tags). Clean tags of all samples were clustered (the similarity was 97% by default) to generate operational taxonomic units (OTUs); singleton OTUs and chimeras were simultaneously removed. To obtain annotated species information, each OTUs representative sequence was compared against SILVA (16s), RDP (16s), and Greengenes (16s) databases using usearch-sintax(version v11, San Francisco, USA).

Fresh feces from mice in the untreated group were collected and immediately re-suspended in aseptic saline; insoluble particles were filtered out. The filtrate was centrifuged at 4 °C for 15 min at 3000 $\times$ g, and the sediment collected. The sediment was dissolved in sterile saline to a final concentration of 400 mg/mL. Each mouse was treated with intragastric administration of 200 $\mu$L per day [14].

Influenza A virus (A/FM1/1/47) was provided by the Department of Immunology and Microbiology at the Jinan University and stored at –80 °C. After mice were anesthetized via intraperitoneal injection of 1% pentobarbital sodium, each was given 50 $\mu$L of virus solution for nasal infection.

The status, coat color, vitality, drinking water, and survival status of mice in each group were observed daily. The body weight of each mouse was recorded at the same time each day, and the percentage of weight change was calculated according to the formula: percentage of weight change = (nth celestial body weight/day 0 body weight). Lung tissue sample was obtained and rinsed with aseptic phosphate buffer (PBS); the surface water was dried with filter paper before weighing. Lung index was calculated as (lung weight/body weight) $\times$ 100%.

Lung tissue and cecum of mice were removed, fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 4 $\mu$m thick sections. Slices were dewaxed, dehydrated, and sealed following hematoxylin-eosin (HE) staining. Finally, histopathological images were observed and collected under a microscope. The histopathological lesion score of each mouse is presented as the average of the scores provided by the two pathologists. The scoring criteria were changed as previously described [15]. so that 0 indicates no lesions, 1–2 indicates mild inflammation with no necrosis, 3–6 indicates moderate inflammation with a high incidence of necrotic cells, and 7–9 indicates severe inflammation with frequent necrosis and edema.

On day 6 after infection (day 32), lung tissue from mice was homogenized in 1 mL of Dulbecco’s Modified Eagle’s Medium (DMEM), and the supernatant obtained using centrifugation. Supernatant of lung tissue was diluted 10 times and to different concentrations. The Madin–Darby canine kidney (MDCK) cell plaque assay was used to determine viral titer in the supernatant of lung tissue. MDCK cells were cultured in a monolayer on a cell culture plate. Following full growth, after each well was washed with PBS, the supernatant of lung tissue with different dilution times was added to each well and incubated at 37 °C for 1 hr. A mixture of 2% carboxymethyl fiber and medium was added and incubated at 37 °C and 5% CO${}_2$ for 4 days. The sample was fixed in 4% paraformaldehyde for 1 h, stained with crystal violet for 15 min, rinsed with pure water to remove excess dye, and the plaques counted. Plaque formation unit (PFU/mL) = (number of plaques per pore / inoculation per pore) $\times$ dilution [16].

Total RNA was extracted from lung tissue homogenate using RNAiso plus (Takara, Kusatsu, Japan). After testing RNA quality, the PrimeScriptTM RT kit and SYBR Green PCR Master Mix (Takara, Kusatsu, Japan) were used for cDNA synthesis and qPCR detection. Reverse transcription was performed in a Bio-Rad S1000 thermal cycler (Bio-Rad, Berkeley, CA, USA). qPCR was performed using the CFX Connect real-time PCR detection system (Bio-Rad, Berkeley, CA, USA). Experimental procedures were as follows: 95 °C, 30 s, 5 s, 60 °C, 30 s, cycle 40 amplification cycles, 95 °C, 10s. All primers (Table 1) were designed and synthesized by Shanghai Shenggong Bioengineering Co., Ltd (Guangzhou, China). All samples were equipped with 3 multiple holes, and the experiment was repeated three times. GAPDH was used as the internal reference and expression was calculated using the cycle threshold method (2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$) [17].

Mice spleen tissues were washed and homogenized in 1640 medium without serum. Peripheral blood mononuclear cells (PBMCs) were collected using a mouse lymphocyte separation solution (Multi Sciences, Hangzhou, China), according to the manufacturer’s instructions. PBMCs were then suspended in 1640 culture containing 10% fetal bovine serum (FBS), and the cell concentration adjusted to 1 $\times$ 10${}^6$/mL. In order to detect Th1, Th2, and Th17 cells, phorbol 12-myristate 13-acetate (0.05 $\mu$g/mL, PMA), ionomycin (1 $\mu$g/mL), brefeldin A (3 $\mu$g/mL, BFA), and monensin (1.4 $\mu$g/mL) were added according to stimulant and blocker reagent instructions (both purchased from Multi Sciences, Hangzhou, China), and incubated at 37 °C for 5 h in a 5% CO${}_2$ atmosphere. Cells were labeled with anti-CD4, interferon (IFN)-g, interleukin (IL)-4, and IL-17A antibodies. In order to detect T regulatory cells (Treg) cells, anti-CD4 and CD25 antibodies were added and incubated at 4 °C for 30 min; cells were re-suspended in a membrane-breaking solution (fixation/permeabilization concentrate: fixation/permeabilization diluent = 1:3, eBioscience, USA) and incubated at 4 °C for 30 min. After washing with 1X permeabilization buffer (eBioscience, USA), anti-Foxp3 antibody was added and incubated for 30 min at 4 °C without light. Th1, Th2, Th17, and Treg cells were defined as CD4 + IFN-g +, CD4 + IL-4 +, CD4 + IL-17A +, and CD4 + CD25 + Forxp3 + T cells, respectively (All antibodies information was in Table 2). All stained cells were detected using flow cytometry FACS VERSE (Becton Dickinson Biosciences, Franklin Lake, NJ, USA), and the results analyzed using FlowJo v10 (FlowJo, Ashland, OR, USA).

Mouse lung tissue was extracted using RIPA lysis buffer (LOT: 6171803, Multi Sciences, Hangzhou, China), with total protein extracted after homogenization. Total protein was quantified using a BCA protein quantitative kit (LOT: A91041, Multi Sciences, Hangzhou, China). Protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF) membrane sealed with 5% skim milk powder. The membrane was then incubated with rabbit anti-mouse monoclonal antibodies against RIG-I (CST, 4200S, 1:1000), MA VS (CST, 4983S, 1:1000), NF-$\kappa$B p65 (CST, 8242S, 1:1000), and GADPH (CST, 2118S, 1:1000) at 4°C. Cells were then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (LOT: A91053, Multi Sciences, Hangzhou, China). Protein bands were detected using an electrochemiluminescence (ECL) kit (LOT: A90844, MultiSciences, Hangzhou, China), and analyzed using ImageJ software (v1.8.0.112, NIH, Bethesda, USA).

Statistical analysis was performed using GraphPad Prism 8.0 software (Beijing, China). Results were expressed as mean $\pm$ standard deviation (SD). Two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests was executed. A p value of 0.05 was regarded as statistically significant.