The second session of the Ethics Review Committee of the National Institute for Viral Disease Control and Prevention (IVDC) at China Centers for Disease Control and Prevention (CDC) determined that the present study followed the working regulations of ethics review committee of Institute for viral disease control and prevention of Chinese Center for Disease Control and Prevention and therefore approved the study. The legal guardians of the patient involved in this study provided written informed consent to have data/samples from her medical records used in research. All methods were performed in accordance with the relevant guidelines and regulations.
A throat swab sample was collected on a 10-year-old girl diagnosed with varicella at Children's Hospital of Chongqing Medical University. The date of onset was estimated on December 21st 2018. The patient immediately found a rash all over her body after having contact with her sister with chickenpox. The patient began to exhibit macular papules that gradually turned into herpetic rash with itching, oral pain and no salivation. During the course of the disease, there was neither fever, spasm, disturbance of consciousness, headache, vomiting, unstable walking, skin/mucous membrane bleeding, abdominal pain, vomiting nor diarrhea. At the age of 9, the patient was diagnosed with Systemic Lupus Erythematosus (SLE) and Lupus Nephritis (LN) and was treated with glucocorticoids and immunosuppressants. The sample was collected in viral transport medium and stored at -80 °C until use.
Next Generation Sequencing (NGS)
Total DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Germany) then fragmented using the ultrasonicator S220 (Covaris, USA). A sequencing library was generated with the KAPA HyperPlus kit (Roche, Switzerland) and sequenced with the NovaSeq 6000 system (Illumina, USA). The paired-end reads have been deposited in the NCBI Sequencing Read Archive under the accession number PRJNA681411.
NGS analysis pipeline
Sequence quality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Sequences were cleaned up with Trimmomatic 4 (http://www.usadellab.org/cms/index.php?page=trimmomatic). The sequences from the human genome (GRCh38) were depleted using the assembler Burrows-Wheeler Alignment tool (BWA) and Sequence Alignment/Map (SAM) tools 1.9 5,6. The remaining sequences were processed in 2 ways: 1- assembly with SPAdes7 ( http://bioinf.spbau.ru/spades ); 2- mapping against the VZV reference strain Dumas (NC_001348) using BWA and SAMtools 1.9 5,6. The VZV-related sequences were then de novo assembled using Sequencher 5.0 (Genecodes Corp., Ann Arbor, MI, USA). The sequence strategy as well as the number of reads at each step of the analysis pipeline are shown in Fig. S1. The full-length genome sequence was annotated in Artemis 16.0.0 8 based on the Dumas reference genome (NC_001348) and submitted to GenBank (MW316406). The viral strain was named VZV/Chongqing.CHN/2018/V. For convenience, however, the sample is referred in the manuscript as SD14.
VZV sequence analysis
Two hundred thirty-two VZV genomes were downloaded from GenBank. Identical or incomplete sequences were discarded. The remaining 222 genome sequences were compared with the de novo SD14 sequence (Table S1). As previously reported, the genome sequence was analyzed based on 8 regions (A to H), excluding the repeats TRL, IR1, IR2, IR3 and TRS2,9. Sequences were aligned using MAFFT 7.311 10 (http://maf.cbrc.jp/alignment/sofware/). Alignments were analyzed using BioEdit 220.127.116.11 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). 7 single nucleotide polymorphisms (SNPs) found in each region were concatenated. Phylogenetic trees were generated with MEGA 6 12. Neighbor joining (NJ) trees were generated with the maximum composite likelihood nucleotide substitution model 12. The phylogenetic inference was tested using the bootstrap method with 1000 replicate 13. Bootstrap values greater than 70% were indicated. Phylogenetic trees were also generated using the maximum likelihood (ML) method in MEGA 14. SNPs found only in SD14 genome were analyzed in Protein Variation Effect Analyzer (PROVEAN，http://provean.jcvi.org/seq_submit.php) in order to check whether these mutations had any effect on protein function 15.
The paired-end reads have been deposited in the NCBI Sequencing Read Archive under the accession number PRJNA681411. The full-length genome sequence of SD14 strain VZV/Chongqing.CHN/2018/V was submitted to GenBank (MW316406).