82 rodents were captured in the forested areas of Hengdaohezi Town (A, N 44°48′44″, E 129°02′04″) Hailin City, Heilongjiang Province from May 21 to August 22 (Summer Festival) in 2020 were selected as the research samples. The collected samples included 22 Myodes rufocanus (MR), 16 Apodemus peninsulae (AP), 15 Apodemus agrarius (AA), 11 Tamias sibiricus (TS), 10 Sciurus vulgaris (SV), and 8 Cricetulus triton (CT). A total of six samples from the same species were mixed.
Fecal pellets samples were homogenized, diluted in a ratio of 1:10 using PBS, made into a suspension, and vortexed for thorough mixing. The samples were then centrifuged at 2000 rpm for 10 min at 4 °C. Following this, the supernatants were transferred to a fresh tube and centrifuged for 10 min for the complete removal of cell debris, bacterial cells, and other impurities. The supernatants were filtered through a 0.22 μm syringe filter (Jet, Guangzhou, China) and concentrated. The filtrate was centrifuged in an SW55Ti rotor using a Beckman ultracentrifuge at 45,000 rpm for 2 h. The precipitates were re-suspended in PBS and passed through a 0.22 μm syringe filter. The samples were then stored at − 80 ℃ until subsequent analyses.
Viral metagenomics analysis
Sequencing libraries were generated using the NEBNext® Ultra™DNA Library Prep Kit for Illumina (NEB, USA), following the manufacturer's recommendations. Index codes were added to attribute sequences to each sample. Briefly, the DNA samples were fragmented by sonication to a size of 300 bp, and the DNA fragments were then end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing to aid further PCR amplification.
Finally, PCR products were purified (AMPure XP system), and libraries were analyzed for size distribution using an Agilent2100 Bioanalyzer and quantified using real-time PCR. According to the manufacturer's instructions, the clustering of the index-coded samples was performed using a cBot Cluster Generation System. After cluster generation, the library preparations were sequenced on an Illumina HiSeq2500 platform, and paired-end reads were generated.
Species annotation and analyses of abundance
Prinseq software (version 0.20.4) was used to assess the sample data quality, filter the low-quality and repetitive sequences and the rodent genome was the reference genome. Bowtie2 software was used to remove host DNA sequences, and Mira (v4.0.2) software was used to splice and assemble the sequences. The assembled and unassembled sequences were searched against the local NCBI virus database using the BLASTX and BLASTN tools in the BLAST + software package to obtain virus annotations. Geneious version 2019.2.1 was used to predict the ORF of the new annotated virus.
Passage of fecal supernatants in BHK-21 cells
The BHK-21 was used to propagate the fecal suspensions from wild mice and detect viruses according to previously published methods [11, 12]. Briefly, the supernatants of homogenized fecal pellets were filtered through a 0.22 µm syringe filter and inoculated onto the BHK21 cells, followed by incubation for 2 h to allow for virus adsorption. After adding a fresh medium, the cells were incubated at 37 ℃ and monitored daily until 7 days postinfection to develop CPE. The infected cells were blindly transmitted three to six times until CPE appeared. The supernatant harvested from CPE-positive BHK-21 cells was further inoculated on BHK-21 cells to detect the presence of infectious viruses.
Detection of astrovirus in CPE-positive cells
BHK-21 cells were inoculated with the supernatant collected from CPE-positive cells and incubated for 36 h. Real-time fluorescent quantitative reverse transcription PCR (RT-qPCR), immunofluorescence detection (IFA) were used to analyze the presence of astroviruses in the inoculated cells.For RT-qPCR analysis, primers that specifically amplify the target genes of astrovirus were designed based on the assembled contig sequences.
The sequences of reference strains with high similarity to the virus described in this study were downloaded from NCBI. A phylogenetic tree was constructed using MEGA software based on the neighbor-joining maximum composite likelihood method with 1,000 bootstrap replicates to analyze phylogenetic relationships.
MetaStat was used to analyze the top 10 abundant taxonomic sequence tags of the three samples. The differences were considered to be statistically significant when the P-value was less than 0.05.