Patient samples for this study came from The First Affiliated Hospital of Guangzhou University of Chinese Medicine and Guangdong Province Traditional Chinese Medical Hospital. All patients aged between 18-and 75-years-old with fever > 37.5 ℃ for less than 72 h were invited to participate in the study. Anti-dengue IgM and IgG enzyme-linked immunosorbent assay kits were employed to confirm dengue infection (Radke et al., 2012). We obtained 168 serum samples during the dengue outbreak in Guangzhou in 2014.
DENV isolation was conducted from the patients' positive blood samples. To increase virus titer, we first used an Ae. albopictus gut cell line (C6/36) for virus propagation. The C6/36 cell line was cultured in MEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) for cell maintenance. Patient serum was diluted 10-fold in fresh MEM medium. After the addition of 200 μL serum to the C6/36 cells (1.5×105 cells), the cells were incubated at 37 ℃ for 90 min. Then, the medium was replaced with fresh MEM medium with 2% FBS; the cells were maintained at 28 ℃ with 5% CO 2 for 5 to 7 days (until significant cytopathic effects (CPE), due to infection, were present). Simultaneously, 400 μL supernatants were collected for RNA extraction after centrifugation at 4000 rpm. These samples were then stored at -80 ℃ until needed.
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA according to the manufacturer's in structions; then, the RNA was reverse-transcribed into cDNA, at 42 ℃ for 60 min followed by 72 ℃ for 10 min, with reserve transcriptase. Finally, DENV serotyping was carried out by multiplex RT-PCR (Yong et al., 2007).
After cDNA reverse transcription to make the templates, RT-PCR was performed to amplify the envelope (E) gene (1485 bp). The PCR reaction included 0.3 μmol/L each primer, 1 μL Thermococcus kodakaraensis (KOD)-Plus-Neo polymerase (Toyobo Co., Ltd, Osaka, Japan), 5 μL 10× PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs and 2 μL cDNA. The PCR amplification consisted of 40 cycles of 10 s denaturation at 98 ℃ for 30 s, annealing at 58 ℃ for 50 s and extension at 68 ℃ for 50 s, and a final extension at 68 ℃ for 10 min. The amplicons were purified with Centri-Sep columns (Invitrogen, Carlsbad, CA, USA). Double-stranded sequencing of the E gene was performed on an ABI PRISM 3700 Genetic Analyzer (Applied Biosystems Inc, Foster City, CA, USA) using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Inc, Foster City, CA, USA) according to the manufacturer's protocol. The DENV primers used for sequencing were designed based on the DENV-1 standard Hawaii strain (GenBank accession number: KM204119) and are presented in Table 1.
Reaction Primer name Sequence (from 5′ to 3′) Position Amplification DENV E1 Forward TGCCATAGGAACATCCATCAC 863-883 Amplification DENV E2 Reverse TCCCAATGGCTGCTGATAGTC 2495-2462 Sequencing DENV E1 Forward TGCCATAGGAACATCCATCAC 863-883 Sequencing DENV E2 Reverse TCCCAATGGCTGCTGATAGTC 2495-2462 Sequencing DENV E3 CGGAAAAGGTAGCCTAATAACG 1259-1280
Table 1. Primers used for amplifying and sequencing the complete DENV-1 envelope gene
Ultimately, 17 strains causing CPE were observed, and 17 E gene sequences were obtained from the patient samples. We uploaded the sequences to GenBank and received their IDs: the accession numbers for the sequences are provided in Table 2.
Strain Date of occurrence GenBank accession number District GZ/E24 2014-10-31 KT037099 Yuexiu GZ/E25 2014-11-10 KT037100 Yuexiu GZ/E27 2014-11-20 KT037101 Lianshan GZ/E31 2014-11-5 KT037102 Yuexiu GZ/E101 2014-11-25 KT037103 Liwan GZ/E99 2014-10-5 KT037104 Nanhai GZ/E98 2014-10-5 KT037105 Yuexiu GZ/E86 2014-10-23 KT037106 Panyu GZ/E80 2014-10-29 KT037107 Yuexiu GZ/E79 2014-11-3 KT037108 Baiyun GZ/E66 2014-11-9 KT037109 Tianhe GZ/E19 2014-11-20 KT037110 Baiyun GZ/E6 2014-10-25 KT037111 Yuexiu GZ/37 2014-10-31 KT037112 Yuexiu GZ/E9 2014-10-27 KT037113 Yuexiu GZ/E38 2014-11-3 KT037114 Yuexiu GZ/E41 2014-11-9 KT037115 Tianhe
Table 2. Newly isolated DENV-1 strains from Guangzhou during 2014. The GenBank accession numbers and the districts for the strains are shown
All the DENV-1 sequences (as of 2015) were downloaded from GenBank. The 3456 dengue sequences with known collection locations were retained from the 5483 sequences. The acquired and sample sequences were then aligned with ClustalX v. 2.0 software. CD-HIT v. 4.6 was used to reduce the redundancy, with a threshold level of 0.95. One sequence from each cluster was randomly chosen as a representative among sequences that shared a similar isolation location and time. A maximum likelihood (ML) tree consisting of 443 sequences was reconstructed under the generalized time-reversible (GTR) + gamma-distributed (G) + invariant sites (Ⅰ) model in MEGA v. 6.0. We found 160 genotype Ⅰ, 3 genotype Ⅱ, 4 genotype Ⅲ, 101 genotype Ⅳ and 175 genotype Ⅴ sequences.
To identify the genotypes of the sample sequences, a ML tree was reconstructed with 120 sequences belonging to different genotypes and the sample ones: the sample sequences were found to belong to genotypes Ⅰ and Ⅴ. Another complete ML tree of genotype Ⅰ and Ⅴ was reconstructed with the GTR+G+I (genotypes Ⅰ) and GTR+G (genotypes Ⅴ) models, respectively, as described above.
A total of 250 dengue sequences were downloaded from GenBank using the National Center for Biotechnology Information nucleotide BLAST program (as of 2016), which were compared with the study samples. The downloaded and sample sequences were then aligned with ClustalX v.2.0 software and 157 sequences were retained after removing duplicate nucleotide sequences. Bayesian evolutionary analysis of the local cases from 2013-2015 was performed in BEAST v. 1.8.2 software using the Bayesian Markov chain Monte Carlo (MCMC) method. The mean evolutionary rate of the E gene and the time to the most-recent common ancestor (tMRCA) were calculated through the best-fit model of nucleotide substitution that was selected by jModelTest and the relaxed molecular clock (uncorrelated lognormal). The MCMC sample was analyzed with 100 million replications and every sample was 10, 000 generations. The convergence of the data was assessed by TRACER v. 1.6 software with an effective sample size that was greater than 200. The maximum clade credibility (MCC) was conducted with burn-in of 10% by using TreeAnnotator v. 1.8.2 software. The MCC tree was visualized in FigTree v. 1.4.2 software.