Viruses, Cells, and Viral Load Quantification
RV SA11 strain (provided by Dr Kobayashi, Osaka University, Japan) and fetal African green monkey kidney cells (MA104 cells; Cell Resource Center, IBMS, and CAMS/PUMC, Beijing, China) were used in this study. MA104 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Biochemical, Beijing, China) supplemented with 5% calf serum (FBS; GIBCO, Paisley, UK) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in 5% CO2 at 37°C in a humidified incubator. RV strain SA11 was propagated in MA104 cells as in previous studies42. The virus titer was counted as fluorescent focus assay (FFA), and viral load was quantified by QPCR [22].
Total RNA was extracted from the intestinal contents of mice by using RNAiso Plus (Takara Bio Inc, Dalian, China) following the manufacturer’s protocol. The RNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Biochemical, Beijing, China). The cDNA was prepared from 2 μg of RNA using the following primers: forward: 5’-ATCAGCAAACTGACGAAGCG-3’; reverse: 5’- CCAACTTTTCAGCTGTCGCA-3’ (Takara Bio Inc, Dalian, China). In brief, the amplification was performed using a 10-μL volume reaction in a 96-well plate with the following conditions: 1 cycle at 94°C for 30 s, followed by 36 cycles of 94°C for 5 s, 60°C for 30 s, and 72°C for 30 s. The RV RNA copy levels were quantified by comparison with a standard curve generated using ten two-fold serial dilutions of a plasmid containing the RV VP7 gene.
Animal and Experimental Design
Twenty specific-pathogen-free female Kunming (KM) mice were provided by the Laboratory Animal Center at Jinzhou Medical University (Liaoning Province, China). The female mice were paired with male mice upon delivery. The males were removed the next day, and the pregnant females were monitored daily and allowed to deliver at term. The day of birth was recorded as day 1 of life. Litter statistics and the ratio of males/females in each cage were not calculated. One lactating female mouse and her pups were maintained together in individually ventilated autoclaved cages (IVC). Suckling mice were divided into two groups: RV-infected and uninfected groups. Each group contained 10 litters of mice. There were 7–8 mice in each litter. RV-infected groups of suckling mice were orally administered 50 μL of 106 PFU/mL RV strain SA-11, and the uninfected group were orally administered 50 μL of phosphate-buffered saline (PBS) as a control. All experimental groups were housed in the same specific-pathogen-free room, which was maintained on a 12-h light/dark cycle at 22 ± 2°C with 40–70% humidity.
All of the suckling mice were euthanized four days (the time point at which the most severe diarrhea symptoms presented) post-RV infection. Mixed intestinal contents of the colon and rectum were collected using autoclaved tweezers and stored in sterile tubes at −80°C. All animal experiments were performed in accordance with the Jinzhou Medical University guidelines. All of the animal experiments in this study were approved by the Animal Welfare and Ethical Review Board at Jinzhou Medical University (approval ID: 2019014). All animal infections and infectious work were performed in biosafety level 2 facilities.
Histopathology
Five sucking mice were randomly selected from each RV group and NC group for sample collection and pathological analysis. The duodenum was harvested from the abdominal cavity immediately after the suckling mice were euthanized. For the histopathological investigation, the duodenum was fixed in a solution of 4% paraformaldehyde (0.01 M PBS, pH 7.4). For paraffin section preparation, the duodenum was dehydrated with an increasing ethanol gradient, cleared with xylene, and then embedded in wax. Three consecutive paraffin sections (5 μm thick) were used for hematoxylin and eosin (H&E) staining. For each slice, fields were randomly selected.
DNA extraction and PCR amplification
The RV-infected and uninfected control mice groups were euthanize. Each group contained 10 litters of mice, and there were 7–8 mice in each litter. Genomic DNA was extracted from mixed intestinal contents using the QIAamp DNA Stool Mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer protocols. The V3–V4 region of the bacterial 16S ribosomal RNA genes (342F and 806R) was amplified by PCR using primers 341F 5’-CCTACGGGRSGCAGCAG)-3’ and 806R 5’-GGACTACVVGGGTATCTAATC-3’. The PCR protocol used in this study was as follows: 95°C for 3 minutes, followed by 30 cycles at 98°C for 20 s, 58°C for 15 s, 72°C for 20 s, and a final extension at 72°C for 5 minutes. PCR reactions were performed in a 30-μL mixture containing 15 μL of 2× KAPA Library Amplification ReadyMix (Roche-KAPA, Shanghai, China), 1 μL of each primer (10 μM), 50 ng of template DNA, and ddH2O. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions. The resulting library was analyzed with a Thermo NanoDrop 2000 spectrophotometer (ThermoFisher, Shanghai, China) and 2% agarose gel electrophoresis. Once the library passed the quality inspection, it was quantified by Qubit and mixed according to the data requirements of the quantification and normalization of individual PCR products.
Sequencing of 16S rRNA gene amplicons
The PCR products were quantified using Qubit (Invitrogen, Carlsbad, CA, USA), multiplexed at an even concentration, and subjected to 400–450 bp pair-end sequencing. The adaptor was added to the products, and the samples were sequenced on an Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA). The reads were assembled and used for subsequent 16S analysis. The assembled reads were filtered to acquire clean reads. Reads with an average quality value below 20 and number N that was more than 3 was removed, then the sequences spanning the entire V3–V4 amplicon were matched using PANDAseq[23]. Merged sequences with 97% nucleotide sequence identity (97% identity) were binned into operational taxonomic units (OTUs) using UPARSE [24]. Based on the RDP classifier, a representative sequence of each OTU was assigned to a taxonomic level in the RDP database using 0.8 as the minimum confidence threshold [25].
Statistical analysis of the data
Alpha diversity indices, including the number of OTUs, observed species diversity, and Shannon and Simpson indices, were calculated by normalizing the number of clean reads in all samples to 47504 sequences using mother software [26]. Rarefaction curves were analyzed with mothur and plotted using R. A representative sequence was chosen from each OTU by selecting the sequence that had the largest number of hits in the OTUs. The non-parametric Wilcoxon test (Wilcox. Test in R) was performed for each index of alpha diversity. Rank sum test was used to screen the alpha diversity indices with significant differences under different conditions.
Beta diversity was calculated for the normalized OTU table using UniFrac distance matrices [27,28] in order to determine the amount of bacterial diversity shared between the two groups. Principal coordinate analysis (PCoA) of bacterial communities was performed using weighted UniFrac distances based on the presence and absence of OTUs, and the plot was generated using PERMANOVA, which was also performed using weighted UniFrac distance to test for differences in bacterial community composition in the samples from the two groups.
Linear discriminant analysis effect size (LEfSe) analysis was used to determine the features most likely to explain differences between the RV-infected group and healthy control group. Different features with an LDA score were identified [29]. The P value was set at P < 0.05, and the threshold on the logarithmic linear discriminant analysis (LDA) score was 2 [30]. In addition, heatmap analysis was performed to compare the significant differences at the genus level in the RV-infected and uninfected groups. PICRUSt (version 1.0.0, http://picrust.github.com/)[31] was performed to predict the microbial community function of the two groups. Finally, a statistical analysis of the taxonomic and functional profiles (STAMP, version 2.1.3, http://kiwi.cs.dal.ca/Software/STAMP)[32] was used for further exploration in level 2 of the KEGG analysis using Reconstruction of Unobserved States (PICRUSt) analysis.