Viral metagenomics of fecal samples from zoo mammals in China.

In May to September 2022, we collected 1 145 fecal samples from eight mammalian orders housed in zoos across Southwest, Northeast, and Southeast China. The mammalian orders include: Diprotodontia (marsupials), Primates (non-human primates), Carnivora (carnivorous mammals), Artiodactyla (even-toed ungulates), Perissodactyla (odd-toed ungulates), Pilosa (anteaters and sloths), Proboscidea (elephants) and Rodentia (rodents). A minimum of three individual fecal samples per species were analyzed, with sample sizes ranging from three to 66 depending on species availability and ethical permits. No animals exhibited diarrheal or acute symptoms during sampling. Fecal samples were collected using sterile disposable gloves and sterile swabs. After collection, samples were immediately placed in pre-sterilized cryogenic tubes and flash-frozen in 30 mins on dry ice for transport. Fecal samples were pooled into 130 sample pools depending on the animal species based on equal fecal mass per specimen (1 g/sample). For endangered species with limited specimen availability, all available samples were included. And these sample pools were classified into three categories of feeding habits: herbivore, omnivore and carnivore. Samples were homogenized by suspending in ten volumes of phosphate-buffered saline (PBS) and vigorously vortexed for 5 min, following frozen and thawed three times on dry ice. The supernatants were then collected after centrifugation (10 min, 15 000 ×g, 4 °C) and stored at -80 °C until use. Totally, 500 µL of each supernatant was filtered through a 0.45 µm filter (Millipore, USA) to remove eukaryotic and bacterial cell-sized particles. The filtrates enriched in viral particles were treated with DNase and RNase to digest unprotected nucleic acid at 37 °C for 60 min. Then the remaining total nucleic acid was isolated using a QIAamp Viral RNA Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. For library construction, dsDNA was synthesized from RNA and DNA viruses. For RNA viruses, a reverse transcription kit (SuperScript III Reverse Transcriptase, Thermo Fisher, USA) was used for reversely transcribing RNA into cDNA, and then the DNA polymerase I large fragment (Klenow) was added to synthesize the second strand of cDNA (dsDNA). Specifically, 12 µL nucleic acid extracts were added to the reaction system for synthesizing dsDNA from RNA and ssDNA (total reaction system: 20 µL). For ssDNA viruses, ssDNA was converted to dsDNA using the Klenow reaction simultaneously and all the dsDNA products were used to construct libraries. Overall, 130 libraries were then constructed using a Nextera XT DNA Sample Preparation Kit (Illumina, USA) and the quality was inspected using agarose gel electrophoresis and Agilent bioanalyzer 2100 (China). During library preparation amplification was limited to 16 PCR cycles to minimize duplication biases and maintain library complexity. All libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA) with 250 bp paired ends with dual barcoding for each sample pool. Paired-end reads of 250 bp generated by NovaSeq were demultiplexed by sample-specific dual-index barcodes using Illumina bcl2fastq Conversion Software (v.2.20) with default parameters, assigning reads to their original 130 sample pools. An in-house analysis pipeline running on a 32 nodes Linux cluster was utilized to process the data.