3.1 Epidemic trend of dengue in Yunnan of China and neighboring countries
According to statistics from the World Health Organization, from 2013 to 2019, the number of dengue cases in Yunnan of China and neighboring countries remained at a high level. However, after the outbreak of COVID-19 in 2019, the number of dengue cases in neighboring countries in 2020 has not decreased significantly, while the number of dengue cases in Yunnan province has decreased significantly (Figure 1a and 1b). Then, a comparative analysis of the distribution areas of local cases and imported cases found that the main outbreak areas overlapped and were mainly concentrated in Xishuangbanna and Ruili in Yunnan Province, China (Figure 1c and 1d). In addition, the correlation analysis between the number of imported cases and the number of local cases showed that they showed a significant positive correlation (correlation coefficient = 0.936). In particular, since there is no publicly available detailed data, it is impossible to count the number of DENV1~4 isolates in dengue cases from 2013 to 2019. Therefore, this study counted the number of DENV1~4 isolates in Yunnan from 2013 to 2019 in Genbank data and found that the change trend of DENV1~4 strains is consistent with the trend of dengue epidemic (Figure 2a).
3.2 Screening for DENV prevalence in clinical samples
In this study, among the 172 serum samples, 38 (38/172, 22.1%) serum samples were positive for DENV. From which, 13 of 72 (13/72, 18.1%，9/DENV1, 4/DENV2) serum samples were positive in 2017, 25 of 100 (25/100, 25.0%, 12/DENV1, 13/DENV2) serum samples were positive in 2018. From which, the proportion of DENV2 strains detected in dengue cases rose from 30.8% to 52.0% from 2017 to 2018 (Figure 2). This result is consistent with the statistical data of DENV in GenBank. Then, after removing repeated sequences three complete sequence (MH107161–MH107163) and ten E gene sequences (MH107141–MH107150) were obtained. From which, YN/324 and YN/017 isolate from Myanmar travelers, YN/117 isolate from Laos travelers.
3.3 Sequence Analysis
The complete genome sequences and the E gene sequences of these strains in this study were obtained by amplifying overlapping fragments. From which, YN/117 isolate were obtained from a Laos traveler, YN/017 isolate and YN/324 isolate were obtained from Myanmar travelers. Other strains in this study were obtained from local residents. The genome and the ORF of YN/RL isolate include 10735 nucleotides and 10179 nucleotides (3393 AA), respectively. The 5′ and 3′ UTRs of YN/RL strain were 94 and 465 nt in length, respectively. YN/RL isolate lacks 1 nucleotides (79 nt) in the 5′ UTRs, which leads to significant changes in the RNA secondary structure (Supplementary Figures 1a and 2). Interestingly, the genomes of the YN/MH strain and the YN/017 isolate have different lengths. The ORF and 5′ UTRs were 10176 nt (encoding 3391 AA) and 96 nt in length, respectively. The difference is that compared with YN/MH strain and DENV2-SS (New Guinea C) isolate, YN/017 isolate lacks 13 nucleotides (10270 nt -10282 nt) in the 3′ UTRs, which leads to significant changes in the RNA secondary structure (Supplementary Figures 1b and 3). In addition, the E gene of these sequences in this study all contain 1485 nucleotides and encode 495 amino acids.
3.4 Multiple sequence alignment and phylogenetic analysis
In order to perform genetic evolution analysis, the DENV classic strains of various countries or regions (focus on the three countries bordering Yunnan, Myanmar, Laos and Vietnam) were obtained from NCBI as reference sequences. The phylogenetic divergence analysis based on the E gene sequences of DENV1 revealed that YN/033 strain belong to genotype V, which was the first detected in Yunnan after 2013. Other isolates in this study (YN/251, YN/324, YN/336, YN/075, YN/RL) of DENV1 were clustered in a branch representing genotype I. YN/251 and YN/336 were located in one cluster of the ML tree, with the closest relationship to the YN/324 isolate (Yunnan/Vietnam traveler), Vietnam isolate (2010, KY971707) and Thailand isolate (2001, KY586543). It is worth noting that these isolates were continuously detected in Yunnan and neighboring countries (Laos, Myanmar and Vietnam) from 2013 to 2019. YN/RL strain were in another cluster, with the closest relationship to the Laos isolate (2015, MG894873), and China/Zhejiang isolate (2016, KY886977). Similarly, YN/RL isolate was continuously detected in Yunnan, other province (Zhejiang, Guangdong and Hubei) and neighboring countries (Laos, Myanmar and Singapore) from 2013 to 2019. Special, YN/075 isolate is genetically distant from other isolate in this study and has closeted relationship with Guangdong isolate (2010, JN029812) and USA/Hawaii isolate (1944, EU848545, DENV1-SS) (Figure 3a).
Further analyzed the relationship between the evolutionary tree branching and amino acid substitution of DENV1 revealed that amino acid variations at 10 positions determine the evolutionary differences of gene subtypes. From which, V(I)293M result in Genotype I type; and V436I result in Genotype II type; and K120E, E157G and V345I result in Genotype III type; M(V)293I result in Genotype IV type; F339I, A369T, and I439V result in Genotype V type. In additional, genotype 1 of DENV1 can be divided into 4 subgroups based on the amino acid variation (I122V- Genotype- Ib, N52D and V312L- Genotype Ic, I380V- Genotype Id) (Figure 3b and 3c).
Similarly, phylogenetic analyses revealed that these strains in this study belonged to Asia I (YN/002 isolate), Asia II (YN/017 isolate, YN/MH isolate) and Cosmopolitan (YN/011 isolate, YN/117 isolate, YN/020 isolate, YN/197 isolate). YN/020 isolate and YN/197 isolate has close relationship with YN/117 isolate (Yunnan/Laos traveler), Laos isolate (2018, MN44614) and Malaysia isolate (2014, KX452017). Compare with YN/117 isolate, YN/020 isolate and YN/197 isolate. YN/011 isolate were in another cluster of Cosmopolitan type, with the closest relationship to the India isolate (1992, FJ538925). It is worth noting that these isolates of Cosmopolitan type in this study have been detected in Yunnan (include foreign tourists) and multiple countries (Australia, Singapore, Myanmar, Thailand, Laos, Indonesia and Malaysia) from 2014-2019. YN/MH isolate has close relationship with YN/017 isolate (Yunnan/Myanmar traveler) and Papua New Guinea strain (1944, KM204118, DENV2-SS). Specially, YN/002 strain is genetically distant from other isolates in this study and has close relationship with Laos isolate (2018, MN44619) (Figure 4a).
Combined with multiple sequence alignment and evolutionary analysis results revealed that amino acid variations at 8 amino acid variations are related to genetic evolution. D390N result in America type; and E71K H149N and I462V result in Cosmopolitan type; and V491A result in America/Asia type; and G128E result in Asia- I type; and E126K and M492V result in Asia II type (Figure 4b and 4c).
3.5 Homology and amino acid mutation analysis
The comparison of the complete ORF sequences revealed that the YN/RL isolate share 90.5–97.2% identity to the reference sequence of DENV1. Based on nucleotide similarity analysis of the E protein of DENV1, these isolates of DENV1 in the study shared 89.7%–100.0% similarity with the reference sequence of DENV1 (Table S4). Compared with the E protein of DENV1-SS isolate, these isolates of DENV1 in this study has 22 amino acid mutations, of which 8 amino acid mutations had never been reported (Figure 5a). In particular, YN/324 (Yunnan/Myanmar traveler) strain contain 4 amino acid mutations (H89Q, N92A, V91A, C92G) occurred in the first ED II domain of E protein. Compared to the closest China/Hubei strain (2011, KP772252), a total of 7 amino acid mutations occurred in the CDS of the YN/RL strain (Supplementary Figures 4a).
The complete ORF of YN/MH strain shared 91.9%– 93.9% nucleotide identity with the reference sequence of DENV2. Multiple sequence alignment revealed that the E protein strains of DENV2 in this study showed 89.2%–99.9% identity to the reference sequence (Table S5). Compared with the E protein of Papua New Guinea (1944, KM204118, DENV2-SS), these strains of DENV2 in this study has 17 amino acid mutations, of which 3 amino acid mutations had never been reported (Figure 5b). Compared to the closest DENV2-SS strain, a total of 4 amino acid mutations occurred in the CDS of the YN/MH strain and YN/017 strain (Yunnan/Myanmar traveler). The difference is that compared with DENV2-SS strain and the YN/MH strain, YN/017 strain (Yunnan/Myanmar Traveler) has two amino acids mutations in the NS3 protein (Supplementary Figures 4b).
3.6 Recombination analysis
Multiple studies have confirmed that DENV has widespread intra-serotype and inter-serotype recombination. Combined with RDP4 and SimPlot analysis showed that four strains in this study existed intra-serotype recombination. Among them, the genome sequence of YN/075 strain has a single breakpoint, which is composed of the parental strain (Indonesia/Surabaya strain, AB915384, 2013) and the minor strain (Thailand strain, KY586543,2001). Interestingly, the genome sequence of YN/002 strain, YN/011 strain and YN/017 strain all contained multiple breakpoints. In addition, no recombination between different serotypes has been detected (Figure 6).