Retrospective investigation of the origin and epidemiology of the dengue outbreak in Yunnan, China from 2017 to 2018

Since 2013, a dengue epidemic has broken out in Yunnan, China and neighboring countries. However, after the COVID-19 pandemic in 2019, the number of dengue cases decreased significantly. In this retrospective study, epidemiological and genetic diversity characterizations of dengue viruses (DENV) isolated in Yunnan between 2017 and 2018 were performed. The results showed that the dengue outbreak in Yunnan from 2017 to 2018 was mainly caused by DENV1 (genotype I and genotype V) and DENV2 (Asia I, Asia II, and Cosmopolitan). Furthermore, correlation analysis indicated a significant positive correlation between the number of imported and local cases (correlation coefficient = 0.936). Multiple sequence alignment and phylogenetic divergence analysis revealed that the local isolates are closely related to the isolates from Myanmar and Laos. Interestingly, recombination analysis found that the DENV1 and DENV2 isolates in this study had widespread intra-serotype recombination. Taken together, the results of the epidemiological investigation imply that the dengue outbreak in Yunnan was primarily due to imported cases. This study provides a new reference for further investigations on the prevalence and molecular epidemiology of DENV in Yunnan, China.


Since
, a dengue epidemic has broken out in Yunnan, China and neighboring countries. However, after the COVID-pandemic in , the number of dengue cases decreased significantly. In this retrospective study, epidemiological and genetic diversity characterizations of dengue viruses (DENV) isolated in Yunnan between and were performed. The results showed that the dengue outbreak in Yunnan from to was mainly caused by DENV (genotype I and genotype V) and DENV (Asia I, Asia II, and Cosmopolitan). Furthermore, correlation analysis indicated a significant positive correlation between the number of imported and local cases (correlation coe cient = . ). Multiple sequence alignment and phylogenetic divergence analysis revealed that the local isolates are closely related to the isolates from Myanmar and Laos. Interestingly, recombination analysis found that the DENV and DENV isolates in this study had widespread intra-serotype recombination. Taken together, the results of the epidemiological investigation imply that the dengue outbreak in Yunnan was primarily due to imported cases. This study provides a new reference for further investigations on the prevalence and molecular epidemiology of DENV in Yunnan, China.

. Introduction
The dengue virus (DENV) is a highly prevalent mosquito-borne pathogen, which enters the human body through the bite of Aedes aegypti and Aedes albopictus mosquitoes (1). Clinically, DENV leads to dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), causing a serious threat to human life and health (2). The DENV belongs to the Flaviviruses in the family Flaviviridae. It is an enveloped, single-stranded, negative-sense, RNA virus with a genome (∼11.0 kb) containing only one open reading frame (ORF), which encodes for three structural proteins and seven non-structural proteins. In general, DENVs can be grouped into four different serotypes (DENV 1-4) based on the antigenic differences of the E protein, and its serotypes can be further divided into different genotypes. It is worth noting that there is no cross-protection between different serotypes of DENV (3,4). .
The E protein (∼495 amino acids, AA) is the most fully exposed protein on the surface of mature infectious virus particles and contains crucial immunological epitopes associated with virus neutralization. Moreover, the E protein is the main virulence factor that plays an important role in mediating the adsorption of viruses in host cells, membrane fusion, virus assembly, and virus maturation (5). Three domains (EDI, EDII, and EDIII) are formed in the spatial structure based on the determined crystal structure of the E protein. Among these, EDII, which is located at the amino terminus of the E protein, contains a fusion peptide structure that mediates the fusion of the virus and the target cell (fusion loop, 98-110 AA) (6). Meanwhile, EDIII facilitates the adsorption of viruses to target cells and is considered to contain the receptor binding region as well as the most important neutralizing epitope (7)(8)(9).
DENVs have been popular in more than 100 countries, especially in tropical and subtropical regions. About half of the world's population lives in dengue-affected areas, and more than 50 million people get infected every year (10). The global DF epidemic is becoming more and more serious, threatening global public health (11). In recent years, dengue epidemics have broken out in several regions of China, such as in Guangdong, Zhejiang, Yunnan, Henan, and Hunan (12)(13)(14)(15). In particular, the Yunnan Province of China has been reported as endemic for dengue with numerous cases reported annually. In 2008, a dengue epidemic caused by DENV1 occurred in Ruili City, Yunnan Province, China, a region that is adjacent to Myanmar. In 2013 and 2015, large-scale dengue outbreaks occurred in Xishuangbanna, Yunnan Province, China, which is also adjacent to Myanmar and Laos (15,16). From 2015 to 2016, Dengue outbreaks continue to occur in the border areas between China, Laos, Myanmar, and Vietnam (17). Interestingly, after the COVID-19 pandemic in 2019, the number of dengue cases has dropped significantly. However, no relevant reports are available on the dengue prevalence in Yunnan Province from 2017 to 2018, which is crucial to the traceability and epidemiological detection of the dengue epidemic. To explore the relationship between the COVID-19 outbreak and the substantial reduction of dengue cases, it is necessary to study the epidemic trend and characteristics of dengue from 2017 to 2018, before the COVID-19 outbreak. In this report, we investigated the prevalence of DENV in Yunnan Province, China from 2017 to 2018. This study could fill the gap in the epidemiological surveillance of DENV from 2017 to 2018. Moreover, improving the comprehension of the molecular characteristics and evolutionary trends of the circulating DENV strains.
. Materials and methods

. . Ethical considerations
This study was approved by the Yunnan Institute of Parasitic Diseases and Jilin Medical University (No. 2020_GKJJ028). Written informed consent was obtained from all patients and/or legal guardians of the children involved in this study.

. . Sample collection
A total of 172 human serum samples, which were positive for DENV NS1 and negative for DENV IgG, were obtained from the Yunnan Institute of Parasitic Diseases. The samples were stored at −80 • C before further analyses.
. . RNA extraction and real-time polymerase chain reaction (RT-PCR) Total viral RNA was extracted from human serum samples using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech Co., Nanjing) following the manufacturer's protocol. The primer pairs used were designed based on the DENV reference sequence from NCBI ( Supplementary Tables 1-3). The HiScript R II One-Step RT-PCR Kit (Dye Plus) was used for PCR amplification (Vazyme Biotech Co., Nanjing). The 5 mins TA/Blunt-Zero Cloning Kit (Vazyme Biotech Co., Nanjing) was used to construct the recombinant vectors, which were cloned in Escherichia coli (E. coli, DH5α) before sequencing.

. . Data sources and statistical analysis
The dengue reference sequences used in this study were obtained from the NCBI database and counted. The reported cases in Vietnam, Laos, Cambodia, Philippines, Singapore, and Myanmar were collated from the World Health Organization (WHO) Regional Office for the Western Pacific's Institutional Repository for Information Sharing (WPRO IRIS, http://apps. who.int/iris/). The reported cases in Yunnan Province of China were obtained from the National Health Commission of the People's Republic of China (http://www.nhc.gov.cn/). The data were analyzed using the SPSS software.

. . Phylogenetic analysis
The DENV genome sequences and the E gene obtained in this study have been deposited in GenBank under the accession numbers OQ652963-OQ652975. Reference genome sequences for DENV were obtained from NCBI. The sequences were assembled using the SeqMan software (DNASTAR Inc., Madison, Wisconsin, USA) and were aligned using MegAlign (DNASTAR Inc., Madison, Wisconsin, USA), with the Clustal W alignment method for genomic and AA similarity analyses. Phylogenetic trees were constructed using the neighbor-joining (NJ) method and a pdistance model (bootstrap analysis with 1,000 replicates).

Results
. . Epidemic trend of dengue in Yunnan Province of China and neighboring countries According to WHO, the number of dengue cases in Yunnan Province of China and neighboring countries remained at a high level from 2013 to 2019. After the COVID-19 outbreak in 2019, the number of dengue cases in neighboring countries in 2020 has not decreased significantly, but the number of dengue cases in China has dramatically decreased ( Figures 1A, B). A comparative analysis of the distribution areas of local and imported cases revealed that the main outbreak areas overlapped, mainly in Xishuangbanna and Ruili in Yunnan, China ( Figures 1C, D). Additionally, the correlation analysis between the number of imported and the local cases indicated a significant positive correlation (correlation coefficient = 0.936). However, since there are no detailed publicly available data, it is impossible to determine the number of DENV1-4 isolates among the dengue cases from 2013 to 2019. Therefore, this study determined the number of DENV1-4 isolates in Yunnan from 2013 to 2019 using the GenBank data and found that the change in the trend of DENV1-4 strains is consistent with the trend of the dengue epidemic. However, genome sequencing data for DENV in Yunnan Province. China after 2019 are inadequate in the GenBank database (Figure 2A Figure 2). These results are consistent with the statistical data of DENV in GenBank. In the genome sequencing data, the repeated sequences were removed to obtain three complete sequences (OQ652963-OQ652965) and ten E gene sequences (OQ652966-OQ652975). Among these, the YN/324 and YN/017 isolates were from Myanmar travelers, and the YN/117 isolate was from a Laos traveler.

. . Sequence analysis
The genome and the E gene sequences of the strains used in this study were obtained by overlapping polymerase chain reaction.

. . Multiple sequence alignment and phylogenetic analysis
To perform the genetic evolution analysis, the reference sequences of the classic DENV strains from various countries or regions, with a focus on the three countries adjacent to Yunnan Province of China: Myanmar, Laos, and Vietnam, were obtained from NCBI. Intriguingly, the phylogenetic divergence analysis based on the E gene sequences of DENV1 revealed that the YN/033 strain belongs to genotype V, which was first detected in Yunnan   genotype V. The DENV1 genotype 1 can be divided into 4 subgroups based on the AA variation: I122V-genotype Ib, N52D and V312L-genotype Ic, and I380V-genotype Id (Figures 3B, C).

. . Homology and AA mutation analysis
A comparison of the complete ORF sequences revealed that the YN/RL isolate shares 90.5-97.2% identity with the DENV1 reference sequence. Based on nucleotide similarity analysis of the DENV1 E protein, the isolates in the study share 89.7-100.0% similarity with the DENV1 reference sequence (Supplementary Table 4). Compared to the DENV1-SS E protein, the DENV1 isolates in this study have 22 AA mutations, of which eight AA mutations have never been reported ( Figure 5A) Figure 5B). Compared to the closest DENV2-SS strain, a total of four AA mutations occurred in the CDS of the YN/MH strain and the YN/017 strain (Yunnan/Myanmar traveler). The difference is that compared with the DENV2-SS and the YN/MH strains, the YN/017 strain (Yunnan/Myanmar Traveler) has two AA mutations in the NS3 protein.

. . Recombination analysis
Multiple studies have confirmed that DENV has a widespread intra-and inter-serotype recombination. The combined results of the RDP4 and SimPlot analyses showed that four strains in this study existed via intra-serotype recombination. Among these, the genome sequence of the YN/075 strain has a single breakpoint, which is composed of the major strain (Indonesia/Surabaya strain, AB915384, 2013) and the minor strain (Thailand strain, KY586543, 2001). Interestingly, the genome sequence of the YN/002, YN/011, and YN/017 strains all contained multiple breakpoints. Moreover, no recombination between different serotypes has been detected ( Figure 6).

. Discussion
Yunnan Province of China has a subtropical and tropical monsoon climate with numerous flora and fauna. The southern part of Yunnan Province of China, such as the Xishuangbanna area, is located on the northern edge of the tropics with lush vegetation. The annual average temperature in the area is maintained between 18.9 and 22.6 • C, which is very suitable for the growth and reproduction of mosquitoes. Moreover, Yunnan has close economic exchanges with Southeast Asian countries and is also a popular holiday and tourist destination. This has resulted in very high population mobility in the border areas of Yunnan, which facilitated the spread of mosquito-borne viruses such as DENV, Japanese encephalitis, chikungunya, and Zika (18, 19).
Since Liu et al. first reported the detection of DENV in Yunnan Province in 2006, large-scale dengue outbreaks have occurred in Yunnan every 2 years since 2013, involving different DENV serotypes (20). More specifically, DENV1 was the main serotype that caused dengue epidemics in 2013-2016, DENV2 was mainly involved in 2015, DENV3 was mainly concentrated in 2013 and 2015, while DENV4 was only scattered sporadically (21)(22)(23). Notably, the dengue outbreak areas, such as Jinghong, Mengla, Ruili, Jiangcheng, and Menglian, were mainly focused on border port cities with Myanmar, Vietnam, and Laos. In other words, Yunnan was a province that borders dengueendemic Southeast Asian countries. In the past, it was generally believed that the dengue outbreak in Yunnan was caused by local cases spreading through mosquito bites or mosquitoes spreading across borders. In response to the COVID-19 pandemic, China implemented strict restrictions on cross-border travel to prevent disease spread. As a result, the cross-border floating population has decreased significantly. Surprisingly, the number of dengue cases also decreased from 6,840 in 2019 to 260 in 2020. On the contrary, the number of dengue cases in the Southeast Asian dengueendemic countries adjacent to Yunnan did not drop significantly. Subsequently, a correlation analysis of the regional distribution and trends of local and imported cases revealed a significant positive correlation. The results indicated that the size of the cross-border floating population in Yunnan Province is related to the dengue outbreak. In a study that further explored the sources of the dengue outbreak in Yunnan of China, the results of homology comparison and genetic evolution analysis revealed that there was a high homology and close relationship between the local strains and the Frontiers in Veterinary Science frontiersin.org . /fvets. .     (24). It was concluded that Yunnan is a regional sink for the DENV lineage movement and that the dengue incidence between 2013 and 2019 in Yunnan was closely linked with the international importation of cases. These findings are consistent with our results. Interestingly, the positivity rate of DENV2 has significantly increased from 2017 to 2018 in Yunnan, China. Statistics show that before 2015, DENV1 was the main prevalent serotype in the world. However, since 2015, the scale and frequency of dengue epidemics caused by DENV2 in various countries and regions have been increasing. DENV2 has maintained a high infection rate in Yunnan since 2014. Moreover, in 2015 and 2018, the number of DENV2 dengue cases far exceeded other serotypes. These results suggest that DENV2 and DENV1 may alternately become the main epidemic serotypes in Yunnan Province, China. Genetic evolution analysis revealed that the DENV strains circulating in Yunnan were mainly genotype 1 of DENV1 and Cosmopolitan type of DENV2. It is particularly important to note that some strains have existed stably in Yunnan and neighboring countries for a long time.  (26). In the present study, compared with the DENV1-SS or DENV2-SS isolates, the YN/RL and YN/MH isolates were shown to have multiple base deletions or mutations at the 5' and 3'-UTRs. Specifically, the YN/017 isolate has 13 nts (10,270-10,282 nt) deletions in the 3 ′ UTR. These nt changes led to significant changes in the RNA secondary structure. Whether these changes relate to the level of replication and translation of the isolates remains to be confirmed by further research. Herein, the prM protein could promote the maturation of the virus and increase its infectivity, as well as induce the production of protective antibodies. The E protein contains immunologically important epitopes associated with virus neutralization and has become a potential target protein for vaccine and neutralizing antibody therapy. Deng (29). In the present research, multiple AA mutations were found on the E protein of these isolates. In particular, 12 AA mutations were unique to the isolates in this study and have never been reported. Furthermore, a single mutation (S139N) in the PrM protein of YN/ML and YN/017 isolates was found. Thus, AA substitution in both prM/E proteins may greatly affect the immunogenicity and receptor affinity of DENVs. It is worth noting that the E genes of these isolates have extensive intra-serotype recombination, which can accelerate the mutation and evolution of the virus.
In this study, these isolates also showed multiple AA mutations in non-structural proteins, mainly in NS3 and NS5. Non-structural proteins also act in virus replication and evasion of host immune responses. For instance, Ye et al. indicated that a single silent mutation G66A in the NS2A gene of JEV abolished the production of NS1 in vitro and reduced neurovirulence and neuroinvasiveness in mice (30). The NS3 is a multifunctional protein with helicase, RNA-stimulated nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities. Meanwhile, NS5 is responsible for the replication of the viral genome, RNA capping, and suppression of host interferon responses (31). Thus, AA substitutions in non-structural proteins may greatly affect the efficiency of viral replication.
From 2013 to 2020, Yunnan had many large-scale dengue outbreaks. However, multiple studies have been divergent on the source and prevalence of dengue in Yunnan. This study systematically analyzed dengue epidemic trends, geographical distribution, and genetic evolution in Yunnan and surrounding countries. Multiple pieces of evidence imply that the dengue outbreak in Yunnan was mainly caused by imported cases. Moreover, DENV2 and DENV1 may alternately become the main epidemic serotypes in Yunnan Province, China. Although the prevalence of dengue fever in China has shown a downward trend in recent years, there is still the possibility of repeated epidemics and outbreaks of dengue fever. These could seriously affect the public health system and cause serious economic losses. Therefore, the prevention, control, and monitoring of dengue should still be a priority. We hope that our findings could serve as a reference for future studies on the tracing, epidemic trend, and variation of DENV in Yunnan province, China.

Data availability statement
The data presented in the study are deposited in the GenBank repository, accession numbers OQ652963-OQ652975.

Ethics statement
Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.