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"The investigators used full personal protective equipment during the sampling in the market. Commercially obtained swabs and virus preservation solution were used for the sampling (Disposable Virus Sampling Tube, V5-S-25, Shen Zhen Zi Jian Biotechnology). For environmental samples, sampling swabs were used to swab the floors, walls or surfaces of objects and then preserved in virus preservation solution.
For animal samples, depending on the type of animal and whether it was alive or frozen, pharyngeal, anal, body surface and body cavity swabs or tissue samples were collected for RT-qPCR. Generally, for live animals and frozen full bodies, three samples, including pharyngeal, anal and body surface swabs, were collected for each individual animal. For animal bodies after ‘bai tiao’ preparation (remaining parts of poultry or livestock after removal of hair and viscera), body cavity swabs were collected.
Drain samples were collected using virus sampling swabs to probe into the silt at the bottom of drainage channels in the market. Wastewater and silt samples were preserved in virus preservation solution. For the sewage well (for the drain water), a container was used to take a silt–water mixture from a location near the bottom of the well, and an appropriate amount of sample was collected by using virus sampling swabs and then preserved in virus preservation solution.
Nucleic acid extraction and SARS-CoV-2 RT-qPCR assay
A virus nucleic acid extraction kit (Xi’an Tianlong) was used to extract viral nucleic acid from samples using an automated nucleic acid extraction instrument according to the manufacturer’s instructions. RT-qPCR was carried out on extracted nucleic acid samples with a SARS-CoV-2 nucleic acid assay kit. The reagent brands used include BioGerm (40/38; cycle number/cutoff value), DAAN (45/40) and BGI (40/38).
Virus isolation
Virus isolation was carried out in a biosafety level-3 laboratory in the National Institute for Viral Diseases Control and Prevention, China CDC. Samples positive for SARS-CoV-2 RT-qPCR collected on 1 January 2020 were cultured in both Vero E6 and Huh7.5 cells on 11 January 2020. The cells were cultured in 24-well cell culture plates with DMEM basal medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator containing 5% CO2. Homogenate supernatant was inoculated when the monolayer cell culture was about 90% confluent and adherent to the wall. The medium used was DMEM basal medium containing 2% fetal bovine serum. Three blind passages were carried out for each sample. The growth and morphological changes of the cells were observed under a microscope every day. The culture supernatant and cell pellet of each passage were collected for RT-qPCR. The morphology of viral particles in the cell sections and the supernatant were firstly observed by transmission electron microscopy, on 22 January 2020.
Metagenomic sequencing
Metagenomic sequencing was conducted at the National Institute for Viral Disease Control and Prevention, China CDC and Wuhan BGI. Nucleic acid was extracted using Qiagen’s viral RNA microextraction kit. An enrichment kit (HWTS-C002-16-BGI, BGI, China) was used on 33 samples to improve the sensitivity of viral RNA detection. The kit is based on a probe pool that targets the human ribosomal RNA sequence. The probe pool comprises multiple oligonucleotide fragments, and viral RNA enrichment is accomplished through a sequence of steps including probe hybridization, RNAse H digestion, DNAse I digestion and magnetic bead purification. This specific treatment was chosen based on the low CT values (<30) of internal control (human genes) observed in these samples, indicating a relatively high abundance of human genes. However, the remaining samples did not undergo this treatment. Extracted RNA was reverse transcribed into cDNA and segmented into 150–200 base pairs by enzyme digestion. After repair, fitting, purification, PCR amplification and purification, the sample concentration was assayed by DNBSEQ-T7, and an average output of more than 200 million reads was obtained. Sequencing data were compared with those in a SARS-CoV-2 database to determine whether the samples contained SARS-CoV-2 sequences. For the seven complete SARS-CoV-2 genome sequences, three sequences from environmental samples (Env_0020_seq01, Env_0313_seq02 and Env_0354_seq03) were obtained from DNBSEQ-T7, and four sequences from cell supernatants of Env_0313, Env_0354 and Env_0126 (Fig. 3) were obtained from the NextSeq 550 platform. A few samples were resequenced using a multiplex PCR approach, including Env_0020_seq01, Env_0313_seq04, Env_0313_seq05, Env_0126_seq06 and Env_0354_seq07 (Supplementary Tables 3 and 4), as described previously30. Briefly, the nucleic acid was extracted using Qiagen’s viral RNA microextraction kit. The multiplex PCR comprised a set of 102 oligonucleotide primer pairs and the amplicons generated by the primer pairs spanned the target genome. All raw data related to the genomes, including any partial genomes that were sequenced, were fully reported and deposited to the public database (Supplementary Tables 3 and 4).
Virus genome assembly and phylogenetic analysis
Raw reads were adaptor- and quality-trimmed with the Fastp (version 0.20.0) program. The clean reads were mapped to the SARS-CoV-2 reference genome (GenBank: NC_045512) using Bowtie2. The assembled genomes were merged and checked using Geneious (version 11.1.5) (https://www.geneious.com). The coverage and depth of genomes were calculated with SAMtools (version 1.10) based on SAM files from Bowtie2.
Reference genomes, IVDC-HB-01 (Global Initiative on Sharing All Influenza Data: EPI_ISL_402119) and Wuhan-Hu-1 (GenBank: NC_045512), were used as a query. Multiple sequence alignment of the SARS-CoV-2 sequences obtained from this study and reference sequences were carried out with Mafft (v7.450). Phylogenetic analyses were carried out using RAxML v8.2.9 with 1,000 bootstrap replicates, using the GTR nucleotide substitution model and the Gamma distribution.
Bioinformatic analysis of the species abundances
Kraken2 (version 2.1.2)31 was used for species classification with the option --confidence 0.1. Sequences of all species in the Nucleotide (nt) database were used for generating the index. bracken (version 2.5) was used for re-evaluating species abundance. The matrix of species was obtained by using the pavian algorithm32. The ggplot2 package in R was used for plotting. Read counts of each genus were used for further analysis and plotting. Raw counts for four domains (Archaea, viruses, Eukarya and Bacteria), SARS-CoV-2 and the Homo genus were used to generate a heatmap (Fig. 4b). Two-tailed unpaired t-test was used for identification of differential genus between SARS-CoV-2 RT-qPCR-positive and SARS-CoV-2 RT-qPCR-negative samples.
For the analysis of the Chordata genus characterization, the reference was generated using the sequence of mitochondrial cytochrome c oxidase subunit I in the BOLD system33,34,35. RNA-seq samples were mapped to the reference sequences by the Bowtie2 (ref. 36) algorithm with the default settings. Read counts of each genus were calculated by samtools37. Read counts exceeding 20 were used as a cutoff for the identification of positively enriched genus. Fisher’s exact test was used for comparing the differential genus in the Mammalia class between SARS-CoV-2 RT-qPCR-positive and SARS-CoV-2 RT-qPCR-negative samples.
Ethics
The sample collection was determined by the China CDC to be part of the emergency response to the outbreak of pneumonia of unknown aetiology and therefore was exempt from institutional review board assessment."
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source:
- Article
- Published: 05 April 2023
- William J. Liu,
- Peipei Liu,
- Wenwen Lei,
- Zhiyuan Jia,
- Xiaozhou He,
- Weifeng Shi,
- Yun Tan,
- Shumei Zou,
- Gary Wong,
- Ji Wang,
- Feng Wang,
- Gang Wang,
- Kun Qin,
- Rongbao Gao,
- Jie Zhang,
- Min Li,
- Wenling Xiao,
- Yuanyuan Guo,
- Ziqian Xu,
- Yingze Zhao,
- Jingdong Song,
- Jing Zhang,
- Wei Zhen,
- Wenting Zhou,
- Beiwei Ye,
- Juan Song,
- Mengjie Yang,
- Weimin Zhou,
- Yuting Dai,
- Gang Lu,
- Yuhai Bi,
- Wenjie Tan,
- Jun Han,
- George F. Gao &
- Guizhen Wu
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