Blood serum samples of 54 SFTS patients were collected in 2017 during the acute phase of the disease by the Department of Infectious Disease in Union Hospital (Wuhan City, Hubei Province). Patients were selected according to their clinical manifestations, routine blood tests, and detection of SFTSV by quantitative Reverse Transcription-PCR (RTPCR) using the SFTSV probe qRT-PCR detection kit (Daan Gene, Guangzhou, China) according to the manufacturer's instructions. Demographic features about gender, age, occupation, location, and the disease outcome of all patients enrolled in the study are summarized in Supplementary Table S1. Vero cells (ATCC number: CCL-81) were incubated with each sample to isolate the virus by blind passage, as previously described (Zhang et al. 2017). After three culture passages, indirect immunofluorescence assay (IFA) was performed to detect SFTSV antigen expression in cells and RT-PCR was carried out to detect SFTSV RNA in the cell culture supernatants, as previously described (Zhang et al. 2017). The complete genome of the isolated strains was sequenced using previously described primers and methods (Zhang et al. 2017). Personal information, clinical manifestations, and laboratory test results of each patient from whom SFTSV was isolated are listed in Table 1 and Supplementary Table S2.
Case1 Case2 Case3 Case4 Case5 Case6 Case7 Case8 Case9 Case10 Case11 Personal information Gender Female Female Female Male Male Male Male Female Male Female Male Age 54 43 34 72 69 69 76 47 72 66 45 Location XG SZ HG HG SZ HG HG HG HG SZ HG Occupation Farmer Others Civil servant Farmer Farmer Farmer Farmer Others Others Farmer Others Outcome Survived Survived Survived Fatal Lost at follow-up Survived Fatal Survived Fatal Survived Survived Lab tests Virus Load in serum sample (RNA copies/mL) 1.41 × 104 1.14 × 103 3.69 × 105 < 100 4.83 × 104 < 100 6.94 × 104 1.4 × 103 5.57 × 106 < 100 5.62 × 104 Strains HBXG51 HBSZ52 HBHG54 HBHG8 HBSZ11 HBHG29 HBHG30 HBHG35 HBHG36 HBSZ55 HBHG38 Genotypes L segment C2 C2 C2 C3 C3 C3 C3 C3 C4 C2 C4 M segment C2 C2 C2 C3 C3 C3 C3 C3 C4 C2 C4 S segment C2 C2 C2 C3 C3 C3 C3 C3 C4 C3 C3 XG Xiaogan City, SZ Suizhou City, HG Huanggang City.
Table 1. Personal information, and laboratory tests for SFTS patients.
To analyze SFTSV phylogeny, a dataset was established, including the complete SFTSV coding sequence (CDS) (L, M, and S segments) deposited in GenBank until December 2018 and the new strains reported in this study. In total, 287 L segment sequences, 309 M segment sequences, and 512 S segment sequences were analyzed. The GenBank accession numbers and locations of strains used for the analysis are shown in Supplementary Table S3. Phylogenetic trees were constructed using Mega 6.0, as previously described (Shi et al. 2017). Reassortment events were detected using the RDP software package as previously described (Shi et al. 2017). To analyze SFTSV migration, viral sequences were aligned by using Clustal Omega (https://www.ebi.ac.uk/), and phylogeography trees based on alignment of L, M, and S segments, respectively, were constructed by BEAST 2.3.x (Ancestral Reconstruction/Discrete Phylogeography method).
Pairwise comparison of nucleotide sequences of the RdRp, GP, NSs, and NP ORFs was performed using the Megalin software (Version 7.1) in the DNAStar package. Sequence identity was normalized using the C1 genotype SD4/China/2010 strain (GenBank number: HM802202-HM802204) as the reference. This strain was among the first batch of strains isolated from patients in 2010 when the SFTS disease was first reported (Yu et al. 2011). Sequence identity within each genotype and normalized against the SD4/China/2010 strain are shown using boxplot and violin-plot, respectively. Amino acid (aa) variations of the four SFTSV proteins (RdRp, GP, NP, and NSs) were analyzed by aligning all the protein sequences in the dataset using Clustal W. Variations associated with each SFTSV genotypes were plotted using ggtree and ggplot packages in R Studio (version 3.5.2). The relevant aa positions in the GP 3D structure (PDB code: Gn, 5Y10; Gc, 5G47) were visualized using Pymol (version 4.40).