The Geographic Analysis of Hunan Province and Study Design
The geographic relationships between Hunan Province and the DENV outbreak areas in China were analyzed. The results showed that Hunan had become a central area of the DENV epidemic, which is surrounded by Yunnan, Guangdong, Guangxi, Hainan, Fujian, Zhejiang and the other dengue outbreak areas (Fig. 1) (The map in the figure was drawn by ourselves. Part of the data in the figure is cited from Zhao, et al. [26], and part of the data was provided by Centers for Disease Control and Prevention of Hunan Province).
During the DENV outbreak in Qiyang County, Hunan, from September 2018, a total of 260 serum samples of fever patients were collected, and 96 cases were confirmed to be NS1-positive by colloidal gold testing. Seven strains were proliferated in C6/36 cells for over 6 days to build a viral seeds pool of Hunan DENV. Eighty-nine viral RNAs were successfully extracted directly from 96 NS1-positive serum samples, followed by gene sequencing of the DENV structural protein C/prM/E genes. The phylogenetic analysis, recombination and selection pressure analysis, potential secondary structure prediction based on structural gene sequences originating from epidemic strains were performed to understand the genetic characterization, potential source, and evolution. The study design is shown in Fig. 2.
Phylogenetic analysis
The E protein gene sequences of 123 representative DENV-2 strains and four serotypes of standard strains were selected to construct phylogenetic trees with MEGA software version 7.0. The result showed that all 89 strains in this study were cosmopolitan DENV-2 genotypes. The closest relative was the Zhejiang epidemic strain (MH010629, 2017), followed by strains isolated from Malaysia (KJ806803, 2013), Bali (KT806318, 2014), Indonesia (KT781561, 2014), and the Philippines (KU517847, 2015) (Fig. 3). Among the neighboring provinces of Hunan Province, Zhejiang, Yunnan and Guangdong provinces had more than 1000 reported cases of dengue fever in each province in 2017, and all four serotype have been detected in each provinces [15, 20]. There were also reported cases in Fujian Province in 2017. Since only one amino acid mutation (I431V/A) was observed in all 89 epidemic strains compared with the nearest related strain from Zhejiang (MH010629, 2017). These data suggest that the causative agent of DENV outbreak in Hunan Province in 2018 may come from the epidemic strains in Zhejiang Province in 2017.
Bases and amino acid mutations
Three structural protein-overlapped fragments of epidemic strains were obtained by PCR amplification. After sequencing, the proteins were effectively spliced, and the length of coding nucleotide sequences was 2,325 nt, encoding 775 amino acids. The homology between isolates was 99.7-100%, and the amino acid (AA) sequence of E protein was highly conserved. By comparison, the comparability of nucleotide and amino acid sequences between the 89 epidemic strains and DENV-2SS were 93.5 and 97.8%, respectively. Compared with DENV-2SS, two hundred fifteen bases had mutations in the structural protein region of epidemic strains, among which 195 were synonymous mutations and 20 were non-synonymous mutations, leading to 17 AA substitutions (Fig. 4). Two AA substitutions at 104th (C104: M→I) and 108th (C108: L→M) were observed in protein C in isolate strains, six amino acid mutations including 143th (M29: D→N), 166th (M52: K→N), 196th (M82: T→A), 241th (M127: I→V), 262th (M148: H→Y), and 266th (M152: A→V) occurred in the structural protein prM/M, and nine amino acid mutations including 332th (E52: Q→H), 351th (E:71 D→A), 406th (E126: K→E), 409th (E129: V→I), 429th (E149: H→N), 444th (164: I→V), 602th (E322: I→V), 670th (E390: N→S), 742th (E462: I→V) were observed in structural protein E (Fig. 4).
Potential secondary structure of the structural protein region
The protein secondary structure among DENV-2 standard strain KM204118 and three randomly selected sequences (HNQY2018014, 021, and 028) from the 89 isolate strains were predicted. Compared with DENV-SS, Hunan epidemic strains had missed one nucleotide-binding site (site 6) and one DNA-binding site (site 18), as well as one protein binding region (sites: 4 and 5) in the capsid protein (Fig. S1), while one new DNA-binding site (site 74) and two new protein binding sites (19 and 29) were observed in isolate strains. Moreover, variations were found in the disordered region among Hunan epidemic strains, DENV-2SS and Zhejiang/2017 (Fig. S1). In the prM/M region, which contained 166 amino acids, the protein secondary structure of the epidemic strains was highly consistent with that of the Zhejiang/2017 strain (Fig. S2). However, compared to the DENV-2SS, three protein binding regions disappeared in Hunan epidemic strains (sites: 122, 133, and 220), and one novel protein binding region emerged (site 144). Additionally, one helical transmembrane region of the isolates visibly differed from the DENV-2SS, and eight significant changes were observed in the buried and exposed region, while no noticeable variation was found in the strand and helix region (Fig. S2). Three protein binding sites (sites: 584, 596, and 642) disappeared at the 495AA locus of E protein, one protein binding location (site 377) was observed in Hunan isolates, four considerable alterations were also observed in exposed and buried regions, and minor changes were found in the helical transmembrane and disordered region (Fig. 5). Meanwhile, there were 22 changes in strand regions. Of them, 11 were new (120, 166, 192, 309, 334, 347, 446, 455, 512, 582-584, 591), 11 were missing (101, 102, 124, 141, 207, 290, 294, 553, 607, 636, 651, 692-695), and nearly 70% of changes occurred in E proteins. Nevertheless, compared with the Zhejiang 2017 strain, there was no significant change in protein binding region and polynucleotide-binding region in structural protein (C, prM/M, and E) (Fig. 5, Fig. S1, and Fig. S2).
Possible three-dimensional structure of the structural protein E genes
The possible three-dimensional structure of structural proteins of the representative epidemic strains (HNQY2018014, 021, and 028) were predicted and compared with DENV2-SS and Zhejiang/2017 strain. Homology modeling revealed that five strains had the same three-dimensional structure. In addition, binding sites were also predicted by the 3DLigandSite ligand binding site prediction server, four protein binding sites (HIS429, ALA430, THR435, and GLY436) were observed in DENV-2SS (Fig. 6E). Hunan epidemic strains and the Zhejiang/2017 strain have the same binding sites at ASN429, THR435, and GLY436) (Fig. 6D). HNQY2018028 has two different binding sites (429 and 430) compared to DENV-2SS (Fig. 6) and one diverse binding site (429) compared to Zhejiang/2017.
Recombination and election pressure analysis
RDP4 software was used to analyze potential recombination events among HNQY2018001-HNQY2018089 and other representative DENV-2 virus strains. Preliminary analysis results showed that no recombination event occured in these DENV-2 strains (p<0.05). The structural proteins of 202 strains were analyzed, including 113 representative strains of DENV-2 and 89 isolate strains. The results showed that the MEME method identifies the maximum number of actively selected sites (n = 16). However, the FEL, IFEL and FUBAR methods indicated that all 775 sites were under negative pressure (Table 1). Therefore, no significant evidence of positive selection was presented in at least three different methods, so the positive sites of selection pressure at these sites cannot be determined.