Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2011-2016

A total of 1,596 laboratory-confirmed imported dengue cases were identified in Taiwan during 2011–2016. Most of the imported cases arrived from Southeast Asia as well as the Indian subcontinent, the Pacific region, Latin America, Australia and Africa. Phylogenetic analyses of the complete envelope protein gene sequences from 784 imported dengue virus (DENV) isolates were conducted, and the results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant serotype/genotype of DENV strains circulating in Southeast Asia. The DENV-1 genotype III, DENV-3 genotype III and DENV-4 genotype I and II strains were found to be newly emerging in several Southeast Asian countries. Our results also showed that geographical restrictions of DENV-1 genotype I, DENV-1 genotype III and DENV-2 Cosmopolitan genotype are becoming blurred, indicating the extensive introductions and continuous expansions of DENV strains between nations in Southeast Asia. In this study, we present the geographic distribution and dynamic transmission of DENV strains circulating in Southeast Asian countries. In addition, we demonstrated local dengue epidemics caused by several imported DENV strains in Taiwan during 2011–2016.

Introduction hospital-based reporting system) and active (such as fever screening at airports, self-reporting, and expanded screening for contacts of confirmed cases) surveillance systems were implemented by the central and local health departments in Taiwan. Human serum samples of suspected dengue cases were submitted to the Centers for Disease Control, Taiwan (Taiwan CDC), for confirmation of DENV infection. The human serum samples used in this study were derived from confirmed dengue cases submitted to the Taiwan CDC during 2011-2016. All samples analyzed were anonymized. The study protocol was reviewed and approved by the Taiwan CDC Institutional Review Board (IRB 104121). The informed consent requirement was waived by the board. An imported dengue case was defined as a laboratory-confirmed dengue case with travel history to endemic countries within 14 days before the date of onset of dengue. An indigenous case was recorded when no overseas travel was indicated.

Laboratory diagnosis
DENV infection was defined as a febrile illness associated with the detection of DENV RNA by reverse transcription-polymerase chain reaction (RT-PCR), isolation of DENV by cell culture, detection of DENV nonstructural protein 1 (NS1) antigen, or a seroconversion or at least a four-fold increase in the titer of IgM or IgG antibodies against DENV in paired acute and convalescent serum samples tested by capture IgM and IgG enzyme-linked immunosorbent assays (ELISA). Isolation of DENV was performed using a mosquito cell line (clone C6/36 of Ae. albopictus cells) as previously described [18]. Briefly, for each acute-phase serum sample, 50 μL of the sample diluted at ratios of 1:20, 1:40, 1:80, and 1:160 with RPMI 1640 medium (Gibco/BRL, Life Technologies, Auckland, New Zealand) containing 1% fetal calf serum was added to a 96-well microtiter plate. Then, 1x10 5 cells/100 μL/well of C6/36 were added to the microtiter plate and incubated for 7 days at 28˚C. Cells were harvested, and infection was confirmed by immunofluorescence assay using dengue serotype-specific monoclonal antibodies, including 5F3-1 (DENV-1 specific, ATCC HB-47), 3H5-1 (DENV-2 specific, ATCC HB-46), 5D4-11 (DENV-3-specific, ATCC HB-49) and 1H10-6 (DENV-4-specific, ATCC HB-48). The viruses were subcultured in C6/36 cells and harvested for nucleotide sequencing after the first or second passage. Isolated viruses were identified using the nomenclature of serotype/country of origin/strain/year of isolation. To detect and differentiate DENV serotypes in acute-phase samples, we performed one-step, SYBR Green I-based, real-time RT-PCR (QuantiTect SYBR Green RT-PCR kit, Qiagen, Hilden, Germany) using the LightCycler 96 Real-Time PCR System (Roche Diagnostics, Mannheim Germany). Real-time RT-PCR was performed using two sets of consensus primers, one primer set targeting a region of the nonstructural protein 5 (NS5) genes to detect all of the flaviviruses and the other primer set targeting a region of the C gene to detect all of the DENV serotypes. The DENV serotypes of the positive samples were then confirmed by DENV serotyping using four sets of serotype-specific primers targeting the C gene [20]. A commercial DENV NS1 Ag strip rapid test kit (Bio-Rad Laboratories, Marnes La Coquette, France) and SD Dengue NS1 Ag test (Standard Diagnostics, Inc. Kyonggi-do, Korea) were used to detect the DENV NS1 antigen in serum samples. Envelope (E)/Membrane (M)-specific capture IgM and IgG ELISA were used to detect DENV-specific IgM and IgG antibodies as previously described [21].

Preparation of viral RNA, RT-PCR amplification and nucleotide sequencing
Viral RNA was extracted from acute-phase serum samples or the culture supernatant of C6/36 cells infected with each of the isolated DENV strains using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Primers used for amplification and sequencing of C, prM and E gene sequences of DENVs were described previously [17] [18]. The RT-PCR reaction was carried out with the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen). The cDNA synthesis step was performed at 55˚C for 30 min; PCR at 94˚C for 2 min; 40 cycles of 94˚C for 15 sec, 50˚C for 30 sec, and 68˚C for 1 min; and prolonged elongation at 68˚C for 5 min. PCR products were purified using a Qiagen QIA quick Gel Extraction Kit (QIAGEN). Nucleotide sequences were determined by an automated DNA sequencing kit and an ABI Prism 3730XL DNA sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer's protocols. Overlapping nucleotide sequences were combined for analysis and edited with the Lasergene software package (DNASTAR Inc, Madison, WI). Nucleotide sequences of the complete E gene of the DENV strains described in this study were submitted to GenBank with the following accession numbers: 334 DENV-1 strains (KT175076-KT1750 78, KT175082-KT175101, KT175103-KT175110, KU365900, KY496854, KY496855, and MG89 4671-MG894970), 234 DENV-2 strains (KT175111-KT175140, KU365901, and MG894971-MG895173), 133 DENV-3 strains (KP176703-KP176710, KP175715, MG895174-MG895297), and 99 DENV-4 strains (MG895298-MG895396). All the strain identifiers and their accession numbers are shown in the S1 Table.

Phylogenetic analysis
The nucleotide sequences of the complete E gene of 784 imported and 16 epidemic strains in Taiwan in combination with sequences of epidemic strains from Southeast Asian countries and various global reference strains of different genotypes available from GenBank were analyzed. In addition, sequences representing the most closely related to the epidemic strains in Taiwan obtained using BLAST were selected for phylogenetic analyses. Sequences of DENV strains were aligned, edited and analyzed using Clustal W software [22]. The phylogenetic analysis was performed using MEGA version 7 (http://www.megasoftware.net/) [23]. To construct the phylogenetic trees, the maximum likelihood method using the general time reversible as a substitution model and the neighbor-joining method using the maximum composite likelihood as a substitution model were utilized. The reliability of the analysis was evaluated by a bootstrap test with 1,000 replications. Sequences of D2/New Guinea/NGC/1944 strain/ M29095, D2/Senegal/DAKHD10674/1970/AF231720, D1/USA/Hawaii/1945 strain/AF425619 and D2/New Guinea/NGC/1944 strain/M29095, were used as outgroups to root the tree of the DENV-1, DENV-2, DENV-3 and DENV-4 strains, respectively.

Imported dengue cases in Taiwan during 2011-2016
A total of 1,596 laboratory-confirmed imported dengue cases (both visitors to Taiwan and local returning residents) were identified in Taiwan during 2011-2016. Among them, 703 cases (44.0%) were identified by fever screening at airports (Table 1) and most (>90%) of these cases were in their viremic stages with positive real-time RT-PCR and negative IgM and IgG results. Most cases arrived from Southeast Asian countries, with Indonesia (24.8%, 396 cases), the Philippines (19.2%, 306 cases), Malaysia (14.2%, 226 cases), Thailand (12.0%, 192 cases), and Vietnam (12.0%, 191 cases) being the most frequent country sources of importation. Cases were also imported from other Asian countries (16.6%, 265 cases, including Myanmar, Singapore, Cambodia, India, China, Bangladesh, Maldives, Sri Lanka, Laos, Saudi Arabia, and Japan), the Pacific region (0.7%, 11 cases, including Palau, Papua New Guinea, Nauru, Fiji, Solomon Islands, Tuvalu, and French Polynesia), Australia (0.1%, 2 cases), Latin America (0.4%, 7 cases, including Brazil, Costa Rica, and Saint Lucia), and Africa (0.1%, 2 cases, one from South Africa and the other from Kenya). Comparing the numbers of imported dengue cases between 2003-2010 and 2011-2016, we found there is an increasing trend of imported cases from the Philippines, Malaysia and Singapore during the study period.

Genotype distributions of imported DENV strains
Among the 1,596 imported dengue cases, 784 DENV strains were isolated from acute-phase serum samples of patients infected in 23 countries (Table 1). Phylogenetic analyses of the E    (I, II and III). Genotype I contains the majority of strains from Asia, while genotype II comprises a smaller set of Asian and Pacific strains. Genotype III contains viruses from a wide geographic area [24]. Genotype I of DENV-1 was the predominant genotype imported from Southeast Asian countries, including Indonesia, Malaysia, Vietnam, Thailand, Myanmar and Cambodia. Before 2006, genotype II of imported DENV-1 was the main genotype in Indonesia; however, since 2007, the main genotype has shifted to genotype I. Genotype II was the predominant genotype of imported DENV-1 from the Philippines. The numbers of genotype III of imported DENV-1 strains from Malaysia and Singapore increased during 2013-2014. Fig 5 shows genotype distributions of imported DENV-2 strains. The DENV-2 strains can be classified into six genotypes. The Cosmopolitan genotype has a wide geographic distribution. The Asian genotype 1 and 2 contain viruses from Asia, and the Asian/American genotype comprises viruses from Southeast Asia and Latin America. The American genotype consists of viruses from Latin America and older isolates collected from Indian subcontinent and the Pacific, and the Sylvatic genotype contains sylvatic strains from Asia and Africa [25]. The Cosmopolitan genotype was the predominant genotype of imported DENV-2 strains from Indonesia, the Philippines, Malaysia, and Singapore, and Asian genotype 1 was the main genotype of imported DENV-2 strains from Vietnam, Thailand, Myanmar and Cambodia.  Philippines, the Pacific islands and Australia. Genotype II contains viruses from Southeast Asia. Genotype III has a wide geographical distribution which includes Asia, Africa and Latin America. Genotype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus isolates [26]. Genotype I was the predominant genotype of imported DENV-3 strains from Indonesia and the Philippines. In Malaysia, the number of imported genotype III strains increased between 2015 and 2016. Genotype II was the main genotype of imported DENV from Vietnam, Myanmar and Cambodia. In Thailand, the main detected genotype has shifted from genotype II to genotype III in recent years. Fig 7 shows genotype distributions of imported DENV-4 strains. The DENV-4 strains were separated into four genotypes. Genotype I contains viruses from Asia. Genotype II consists of viruses from Asia, the Pacific and Latin America. Genotype III contains viruses from Thailand and genotype IV contains sylvatic strains from Malaysia [27]. Genotype I was the predominant genotype of imported DENV-4 from Vietnam, Thailand, Myanmar and Cambodia, whereas Genotype II was the main genotype from Indonesia and Malaysia. In the Philippines, the main detected genotype shifted from genotype I during 2003-2009 to genotype II during 2010-2016.
We first made trees for all E gene sequences of the imported and epidemic strains in Taiwan in combination with sequences of epidemic strains from Southeast Asian countries and various global reference strains of different genotypes available from GenBank. In addition, sequences representing the most closely related to the epidemic strains in Taiwan obtained using BLAST

Phylogenetic tree of imported DENV-1 strains
Except for the Philippines, most of the DENV-1 strains isolated from imported cases from Southeast Asian countries belonged to genotype I (Table 1 and Fig 8). Imported DENV-1 genotype I strains from Indonesia and Malaysia showed a high degree of genetic diversity and strains in different lineages that were co-circulating in these countries. Some of the strains from Thailand, Singapore, Myanmar, Laos and China were clustered with strains from Indonesia and Malaysia. Viral strains from Cambodia were closely related to viruses from Vietnam and Thailand, whereas viral strains from Myanmar and Sri Lanka were clustered with viruses from Thailand. Genotype II contained imported viral strains from the Philippines, Malaysia and Indonesia. Genotype III contained imported viral strains from diverse geographical regions, including Asia (Singapore, Bangladesh, Malaysia, Maldives, Thailand, China and India) and the Americas (USA and Costa Rica). It is interesting to note that the strains for DENV-1 tend to be less geographically clustered than in the other serotypes.

Phylogenetic tree of imported DENV-2 strains
The DENV-2 strains isolated from imported cases during 2011-2016 fell into two genotypes, the Cosmopolitan genotype and Asian genotype 1 (Table 1 and Fig 9). The Cosmopolitan genotype strains from imported cases can be divided into three clusters. Cluster 1 contains viral strains from Malaysia, Singapore and Indonesia. Some of the imported viral strains from Thailand, Maldives, Vietnam and China also fell into this cluster. Cluster 2 contains imported viral strains from the Philippines, Tuvalu and Palau. Cluster 3 contains imported strains from India, Saudi Arabia and Kenya. A strain from Thailand and two strains from Vietnam were also found to cluster with strains from India. Asian genotype 1 contains viral strains from Thailand, Vietnam, Cambodia, Lao, and Myanmar. Imported strains from Malaysia also fell into this genotype. No Asian/American genotype and Asian genotype 2 strains were found among imported cases during 2011-2016.

Phylogenetic tree of imported DENV-3 strains
The DENV-3 strains isolated from imported cases fell into three genotypes, genotype I, II and III (Table 1 and Fig 10). Genotype I can be divided into two clusters: one contains viral strains from Indonesia, Malaysia, Singapore and Solomon Islands, and the other contains viral strains from the Philippines. Genotype II contains imported strains from Vietnam, Thailand, Cambodia, and Laos. Genotype III contains viral strains from diverse geographical localities, including India, Singapore, Malaysia, Thailand, Vietnam and Cambodia.

Phylogenetic tree of imported DENV-4 strains
The DENV-4 strains isolated from imported cases fell into two genotypes, genotype I and II (Table 1 and Fig 11). Genotype I contains two major clusters: one cluster contains viral strains from the Philippines, and the other contains viral strains from Vietnam, Thailand, Myanmar and Cambodia. In 2016, an imported strain from the Maldives also fell into this cluster and was closely related to virus strains from Sri Lanka. Genotype II contains imported viral strains from the Philippines, Indonesia, Malaysia and Singapore. Imported viral strains from Papua New Guinea and Brazil also belonged to this genotype.  Notably, genotype III of the DENV-1 strain was found to newly emerge in Malaysia, Vietnam, Thailand and Singapore. In addition, genotype III of the DENV-3 strain also emerged in Malaysia, Thailand and Singapore. However, Asian genotype 2 and the Asian/American genotype of the DENV-2 strain were not found in imported cases from Southeast Asian countries in the last decade, suggesting a low prevalence of these two genotypes in this region.

Major dengue epidemics in Taiwan during 2011-2016
Previous studies have shown that DENV-1 genotype I was the predominant DENV genotype circulating in Southeast Asian countries [18,19,[28][29][30][31][32]. In our study, the numbers of imported DENV-1 genotype I strains increased sharply in Indonesia and Malaysia and became the predominant genotype in the last decade. Phylogenetic analysis of E gene sequences of imported DENV-1 genotype I strains from Indonesia, Malaysia, Thailand and Vietnam showed a high degree of genetic diversity. Interestingly, we found that most of the DENV-1 genotype I strains did not segregate into a distinct clade in each country but that viral strains from Indonesia, Malaysia and a few strains from Singapore, Thailand, and Laos were clustered together. In addition, most of the imported DENV-1 genotype I strains from Thailand, Myanmar, Cambodia, China and Sri Lanka formed another cluster [33][34][35]. A DENV-1 genotype I strain (D1/Laos/1508aTw/2015) isolated from a case imported from Laos in 2015 is closely related to virus strains from Malaysia. Although only a few DENV strains have been isolated from imported cases from Laos in Taiwan during 2003-2016, genotype distribution of these imported DENV strains within each serotype is consistent with the results of a study by Castonguay-Vanier et al. [36]. Genotype III of DENV-1 strains imported from China, Vietnam, Malaysia, Singapore and Bangladesh were clustered together. The results suggest a close genetic relationship and frequent transmission of DENV-1 among Southeast Asian countries and may reflect frequent trade and travel between these countries [37,38].
Genotype distribution of DENV-2 in Southeast Asian countries remains largely unchanged in the last decade. However, it is interesting to note that the number of DENV-2 Cosmopolitan genotype strains imported from Vietnam and Thailand increased and that Asian genotype I strains were found in Malaysia, indicating that these two genotypes of DENV-2 have expanded into new territories. Imported DENV-2 strains from the Maldives during 2015-2016 belonged to the Cosmopolitan genotype and are closely related to virus strains from Malaysia and Singapore. A DENV-2 Cosmopolitan genotype strain (D2/Palau/1612aTw/2016) isolated from a case imported from Palau is closely related to virus strains from the Philippines, Papua New Guinea and Fiji, suggesting cocirculation of these virus strains among these countries.
Except for Malaysia and Thailand, the genotype distribution of DENV-3 strains imported from Southeast Asian countries remains largely unchanged. It is interesting to note that the genotype of DENV-3 strains from Malaysia shifted from genotype I to genotype III. In addition, the genotype of DENV-3 strains from Thailand shifted from genotype II to genotype III in recent years. Recent studies have shown that the DENV-3 genotype III strains are emerging in Asian countries [39][40][41]. In this study, the number of DENV-3 genotype III strains has been increasing in Malaysia and Thailand, and these strains were clustered together with strains from Cambodia, Vietnam and Singapore.
Although a relatively low prevalence of DENV-4 strains was found in Southeast Asia [42], We previously reported that the genetic relationship and genotype distribution of DENV depend largely on the geographical location [18,19]; however, our study demonstrates that geographical restrictions of DENV genotypes are becoming blurred. For example, DENV-1 genotype I, DENV-1 genotype III and DENV-2 Cosmopolitan genotype strains from different geographical locations or countries were clustered together, indicating the extensive introductions and continuous expansions of DENV strains between nations in Southeast Asia.
Due to the small sample size of imported cases from country sources of importation in Taiwan, the potential limitations of our study include: (1) Tourists/travelers may primarily travel to specific locations within a country, and thus, viruses circulating within other regions may not be represented often in our dataset; (2) Potentially changing patterns in travel (i.e. the number of travelers between Taiwan and country sources of importation and the purpose of travel) may affect the numbers of imported dengue cases and the representativeness of our analyses; (3) Because of the limited epidemiological data from country sources of importation, it is unclear whether the serotype/genotype dynamics described in this study are representative of the epidemiological situation within country.
Because air travel has become increasingly popular and convenient, DENV strains will be transmitted by travelers to other countries, sometimes leading to extensive outbreaks. During 2011-2016, several dengue outbreaks occurred in Taiwan, and most epidemic strains were introduced from neighboring Southeast Asian countries, including Indonesia, the Philippines, Vietnam and Myanmar (Table 2). In accordance with our previous studies, the patterns of imported DENV strains observed among the travelers are connected to the overall patterns of the dengue dynamics in Taiwan [19,43]. It is worth noting that an epidemic DENV-1 strain (D1/Taiwan/700TN1109a/2011), which caused the dengue outbreak in southern Taiwan for three consecutive years (2011-2013), is closely related to virus strains from Central America. This is the first time that a dengue outbreak in Taiwan was caused by an American strain and suggests not only that imported DENV strains from neighboring Southeast Asian countries cause local outbreaks but also that viral strains introduced from the Americas or other continents may establish transmission chains within Taiwan.  In this study, we conducted molecular epidemiological analyses to monitor DENV serotype and genotype distributions and dynamic movements in Southeast Asian countries. The DENV strains isolated from imported dengue cases and the availability of a DENV genome sequence database can provide essential information on the global expansion and genetic evolution of DENV, which is useful for disease surveillance, laboratory diagnoses, pathogenesis investigation and vaccine development. Our results also indicate that it is important to reinforce active surveillance and travel and border health measures for dengue prevention and control in Taiwan.  Boldface indicates the same epidemic strains circulated in Taiwan