Background
Dengue is a mosquito-borne disease caused by the dengue virus (DENV), which is transmitted by
Aedes aegypti and
Aedes albopictus mosquitoes that are present in most tropical and sub-tropical regions worldwide [
1,
2]. As a member of the family
Flaviviridae and the genus
Flavivirus, DENV has four distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 [
3]. The disease is prevalent throughout tropical and subtropical regions of the world and is the most rapidly spreading disease, with a 30-fold increase in global incidence over the past 50 years. It is estimated that 390 million dengue infections occur worldwide annually [
4,
5]. Complex factors, including climate change, poor vector control, virus evolution, increased population mobility, uncontrolled urbanization, and the lack of effective vaccines, have aggravated the incidence and geographic expansion of dengue [
6‐
8].
Epidemic dengue outbreaks have been reported in Asia and almost all countries in Southeast Asia, including Laos, Myanmar, Indonesia, Cambodia, Malaysia, Philippines, Thailand, and Vietnam [
9‐
18]. Since the first reported dengue outbreak in 1978 in Foshan, Guangdong Province, China, outbreaks have been reported in Hainan, Guangxi, Guangdong, Fujian, Zhejiang, and Yunnan Provinces [
19‐
24]. Moreover, DENV strains of all four serotypes have been identified in southeastern China.
Yunnan Province, which comprises 16 prefectures and 129 counties, is located in Southwest China. It has tropical and subtropical regions and shares a 4060-km border with its neighboring countries Vietnam, Laos, and Myanmar, all of which are dengue-endemic areas. Since 2000, sporadic imported cases of dengue fever (DF) have been reported almost annually in Yunnan Province, China [
24]. In 2008, a reported epidemic of DF was imported from Mujie City, Myanmar to Ruili City, Yunnan Province, China [
25]. Since 2013, one large-scale dengue outbreak has occurred in Xishuangbanna and Dehong Prefectures of Yunnan Province, which border Laos and Myanmar [
23,
26]. From 2013 to 2015, dengue outbreaks have been reported annually in these areas.
In the current study, we analyzed the phylogenetic, molecular, and epidemiological characteristics of DENV outbreaks and their origins in Yunnan Province that occurred from 2013 to 2015.
Methods
DF case data sources
The case information in this study was retrieved in a blinded manner from datasets preserved at the Xishuangbanna Center for Disease Control and Prevention (CDC), Lincang CDC, and Dehong CDC. The patients’ acute-phase sera (collected within 1–5 days of illness onset) were also obtained from these CDCs. DF case data were obtained from an infectious diseases database report that was officially compiled by the Yunnan Provincial CDC and Chinese CDC. Detailed information was obtained by epidemiologic investigations. Epidemiological characteristics of DF in Yunnan Province during 2013–2015 were descriptive with a retrospective analysis (including temporal and regional distribution, seasonal distribution of dengue cases, demographic distribution, and occupational distribution of DF patients). These data were analyzed by Graphpad Prism v5.0 (GraphPad Software Inc.). Characteristics of age distribution were compared between different age groups using one-way analysis of variance (one-way ANOVA) and Tukey’s multiple comparison test. Results with P values of less than 0.05 were considered statistically significant.
Case definitions and dengue diagnosis
DF was defined according to diagnostic criteria for DF (WS 216–2008) of the Health Department of China. Criteria for clinical-diagnosed DF included a history of mosquito bite and DF symptoms. A laboratory-confirmed case constituted the detection of DENV non-structural protein 1 (NS1) antigen in acute-phase serum. In this study, all DF cases in Xishuangbanna, Dehong, and Lincang Prefectures in 2013–2015 were laboratory-confirmed cases in which DENV NS1 antigen was detected by the Dengue Ag Rapid Test (CTK Biotech, Inc., San Diego, CA, USA).
Serotype identification and amplification of the capsid/premembrane region (CprM) and envelope (E) gene
A total of 232 DENV-positive sera were randomly collected to amplify and sequence the
CprM gene of DENV from patients with DF. Nucleic acids were extracted using an AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, Inc., USA). Reverse transcription PCR (RT-PCR) of viral RNA was performed using the SuperScript® III One-Step RT-PCR System with Platinum®Taq Kit (Invitrogen, Inc., USA). Testing for amplification of the
CprM gene and serotype identification were performed by RT-PCR after RNA extraction using serotype identification primers as previously described by Lanciotti et al. [
27]. The
E gene was amplified with the following primers: DENV1 EF, 5′CAGATACAAAGAGTGGAGACTTGG3′ and DENV1 ER, 5′ATTTGCTTCCACATGATGTTCTC3′; DENV2 EF, 5′TGGATGTCATCAGAAGGGG3′ and DENV2 ER, 5′GGTGTTATTTGTTTCCACATTAG3′; DENV3 EF, 5′AGCTTGGAGACAAGTCGAGAAG3′ and DENV3 ER, 5′CCACAATAGATTCTCCATTCTGG3′; DENV4 EF, 5′AACACCACATTCAGGAATGGG3′ and DENV4 ER, 5′CAGTGAGGTCATGTCCTCCTTC3′.
CprM and
E genes were amplified and cloned into the pMD18T vector (Takara, Inc., China.) for sequencing. DNA was sequenced using the ABI BigDye Terminator Cycle Sequencing protocol on an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA).
DENV isolation
Virus isolation was carried out in Ae. albopictus mosquito C6/36 cells, which were purchased from the ATCC. Acute-phase serum positive for DENV NS1 antigen was diluted 1:20 with Dulbecco’s minimum essential medium (DMEM) (Life Technologies, USA) and inoculated into C6/36 cells for 1–2 h. Inoculated cells were maintained in DMEM supplemented with 100,000 U/mL penicillin, 100 μg/mL streptomycin, and 2% fetal bovine serum (Life Technologies) at 28 °C in 5% CO2. Infected cells were subcultured 3 times and observed daily for the occurrence of cytopathic effects (CPE). Upon CPE in the third subculture, the supernatant was collected and stored at −80 °C until further analysis.
Phylogenetic analysis of isolated DENV
Nucleotide sequences were analyzed using DNAman version 6.0 and compared with sequences available from the BLAST database (
http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model (MEGA, version 6.0;
http://www.megasoftware.net/) [
28]. Bootstrap values ≥70%, calculated from 1000 replicates, are shown at the tree branches. DENV genotype was analyzed by including related reference sequences with known genotypes in the phylogenetic tree [
19,
21‐
23,
26,
29].
Ethics statement
All participants were informed of the aims of the study and the procedures involved in study participation at enrollment; written informed consent was obtained before sample collection. The study was approved by the Institutional Ethical Committee of the Center for Disease Control and Prevention, Chengdu Military Region, the People’s Republic of China.
Discussion
Since 2000, sporadic imported cases of DF have been reported almost annually in Yunnan Province [
24]. In 2008, an epidemic of DF imported from Mujie City, Myanmar was reported in Ruili City, Yunnan Province [
25]. In 2008, a smaller epidemic of 12 cases of indigenous DF occurred in Nansan (Zhengkang County) and Manghai (Mangshi County), which are located in southwestern Yunnan Province and border Myanmar, the suspected source of imported DF cases. In the current study, we confirmed that a large-scale indigenous DF outbreak emerged during 2013–2015 in Jinghong City, Ruili City, and Gengma County, each of which share borders with Laos and Myanmar. In Jinghong City, Xishuangbanna Prefecture, indigenous DF outbreaks of DENV-3 (2013) and DENV-2 (2015) occurred. DENV-1 and DENV-2 emerged annually in Ruili City during 2013–2015, and DENV-4 emerged in 2015. An indigenous DF outbreak of DENV-1 also emerged in Gengma County in 2015.
All dengue infection cases reported in Yunnan Province during 2013–2015 were DF, and not dengue hemorrhagic fever (DHF). In Thailand, DHF was first recognized in 1949 [
30]. Annually, DF/DHF cases are mostly observed in the 5–9-year-old group and the number of cases in the 5–14-year-old group accounts for 70–75% of all reported cases in Thailand [
30,
31]. With respect to the age distribution of dengue cases in Yunnan Province, a comparatively higher incidence (66.40% of all DF patients) of infection was observed in the 20–49-year-old group. In the 0–14-year-old group, the number of cases accounted for 3.89–7.08% of all reported cases in Jinghong City and 8.62–18.15% in Ruili City. This pattern differed from that observed in many dengue-endemic areas in Southeast Asian countries such as Thailand, in which children were most commonly infected [
2,
32]. These results indicated that epidemic dengue in Yunnan Province has emerged in new geographic regions. In 2015, epidemic transmission of dengue in Gengma County occurred in rural villages. However, in Jinghong City and Ruili City, epidemic transmission occurred in large, tropical, urban centers and was part of the urban endemic/epidemic cycle, which is the most important transmission cycle from a public health standpoint [
32].
As four major drivers, failure to control
Ae. aegypti mosquitoes in urban environments, lifestyle changes, urbanization, and globalization have increased the incidence and geographic spread of epidemic dengue [
6]. Yunnan Province contains both tropical and subtropical regions with a 4060-km border with Southeast Asian countries. Previous investigations have confirmed that
Ae. albopictus mosquitoes are widely distributed in the province [
33,
34]; before 2004, however,
Ae. aegypti mosquitoes were not found in this region. In 2004,
Ae. aegypti mosquitoes were found only in the Jiegao Port area of Ruili City; by 2009, investigations of mosquito vectors again confirmed that
Ae. aegypti mosquitoes were distributed locally around Jinghong Port (a port on the Lancang River, also known as the Mekong River) [
35]. These results revealed that
Ae. aegypti has expanded geographically from Southeast Asian countries to ports along the China-Myanmar and China-Laos borders through transport and cargo vessels.
In recent years, population densities and geographic distribution of
Ae. aegypti have increased, especially in urban areas of Yunnan Province [
35]. In 2013, investigations of mosquito vectors by the Xishuangbanna CDC and Ruili CDC revealed that the population densities of
Ae. aegypti are greater than those of
Ae. albopictus. The constituent ratios of
Ae. aegypti and
Ae. albopictus in Ruili City were 82.06% and 18.94%, respectively; in Jinghong City, they were 59.89% and 40.11%, respectively. Comprehensive analysis indicated that the indigenous dengue outbreak in Yunnan Province may be closely related to the expansion and invasion of
Ae. aegypti in this region. Both
Ae. aegypti and
Ae. albopictus can transmit DENV to humans, and their presence in urban areas of Yunnan Province increases the risk of indigenous transmission. In Ruili City, the epidemic period for indigenous cases began earlier and extended longer with each passing year from 2013 to 2015. This may be affected or correlated with meteorological factors such as warming and precipitation enhancement in the rainy season, which has increased mosquito population densities and resulted in failure to control mosquitoes.
The two most common occupational groups identified among DF patients were business and service personnel (30.82%) and housekeepers, unemployed, and retired personnel (22.07%). It is important to note that residents work and live in environments that are densely populated with Ae. aegypti during the day, which increases exposure to infectious mosquito bites and DENV.
The emergence of DHF is associated with introduction of multiple strains of each serotype and the development of hyperendemicity in the region [
6,
36]. Phylogenetic analysis indicated that DENV strains of all four serotypes have been identified in indigenous cases in Yunnan Province. During 2013–2015, DENV-1 genotype I, DENV-2 Asian I genotype, and DENV-4 genotype I were identified in Ruili City. The dengue epidemic pattern observed in Ruili City demonstrated hypoendemic characteristics: circulation of DENV-1 and DENV-2 over consecutive years. Multiple strains of each serotype were introduced, which may increase the risks of DHF emergence in Ruili City. All isolates from Xishuangbanna Prefecture in 2013 were classified as DENV-3 genotype II and isolates in 2015 were classified as DENV-2 Cosmopolitan genotype (or genotype IV). Fortunately, DENV-3 genotype II and DENV-2 Asian I genotype were not detected in dengue cases from Xishuangbanna Prefecture in 2015.
Conclusions
The present study suggested that multiple DENV serotypes are endemic in countries bordering Yunnan Province such as Laos, Thailand, and Myanmar. Imported DF patients from Laos and Myanmar were the primary cause of DF epidemics in Yunnan Province, and Ae. aegypti and Ae. albopictus remain widely distributed in urban regions of the tropics. These factors increase the risk of indigenous dengue transmission and indigenous DF epidemics or pandemics in Yunnan Province. Such evidence indicated that Ruili City is increasingly exhibiting features of an endemic area, as suggested by sustained DENV and the co-existence of multiple serotypes in this area. To prevent future dengue outbreaks and the spread of disease to other regions, joint dengue prevention and control measures should be strengthened in the China-Myanmar and China-Laos border regions of Yunnan Province. In particular, a sound laboratory-based disease surveillance system of imported patients and integrated vector control management are required in both of these regions.