The Geographic Analysis of Hunan Province and Study Design
The geographic relationships between Hunan and the DENV outbreak areas in China were analyzed first. The results showed that Hunan had become a central area of the DENV epidemic, which was 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 map quoted from Zhao , and part 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 from fever patients were collected, and all of these cases were confirmed to be NS1-positive through colloidal gold testing. Of these, 96 DENV-positive serum samples were screened out from patients whose fever courses were shorter than 5 days. Seven strains were amplified in C6/36 cells for over 6 days to construct a viral seed library of Hunan DENV. Eighty-nine viral RNAs were successfully extracted directly from these 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 and the following disposition of study subjects are shown in Fig. 2.
The C/prM/E gene sequences of 89 strains were uploaded to Genbank (http://[email protected]/GenBank/index.html) (GenBank ID: MK543451-MK543470, MK543472-MK543478, MK543480-MK543492, MK949396-MK949438) through Sequin Application (version15.50). One hundred twenty-three different representative DENV-2 strains and four serotypes of standard strains of E protein of dengue virus (DENV) were selected to construct phylogenetic trees with MEGA software version 7.0. The result exhibited that all 89 strains in this study were cosmopolitan DENV-2 genotypes. The closest relative was the Zhejiang strain (MH010629, 2017), followed by strains from Malaysia (KJ806803, 2013), Bali (KT806318, 2014), Indonesia (KT781561, 2014), and the Philippines (KU517847, 2015) (Fig. 3).
Bases and amino acid mutations
Through amplification, three structural protein-overlapped fragments of 89 epidemic strains were obtained. After sequencing, the proteins were effectively spliced, and the length of coding nucleotide sequences was 2,325 nt, 775 amino acids were encoded, 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. Two hundred fifteen bases had mutations in the structural protein region of epidemic strains, among which 195 were synonymous mutations and 20 were nonsynonymous 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 found in structural protein E (Fig. 4). Only one amino acid mutation (I431V/A) was observed in all 89 epidemic strains compared with the nearest related strain from Zhejiang (MH010629, 2017).
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 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 updated in Hunan isolates, four considerable alterations were 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 were observed in DENV-2SS (HIS429, ALA430, THR435, and GLY436) (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 may occur 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 positive sites of selection pressure at these sites cannot be determined.