The current expansion of arbovirus diseases is largely due to mosquito vector range extension, driven by climate change, the globalization of transport, and to increased human movement. The absence of vaccines and efficient therapeutic drugs for the different viruses amplifies the problem. Among arboviruses, Aedes-borne viruses, such as dengue and chikungunya virus, are particularly targeted by national surveillance programs.

Dengue virus (DENV) infects about 390 million people per year worldwide, with 96 million asymptomatic cases [1]. Over the past 50 years dengue has spread inexorably from 9 countries prior to 1970 to over 124 today, with an increase of incidence multiplied by 30 [2]. Vietnam is one of the five countries in the Southeast Asia with the highest dengue burden [3]. First described in northern Vietnam in 1958, dengue expanded in the south during the 1960s [4]. Dengue remains a major health problem in Vietnam and the number of cases has increased over the past 15 years [5]. Vietnam is reported as a hyperendemic country [6–8], with DENV1 and DENV2 being the most predominant serotypes [9]. Dengue transmission occurs throughout the year with peaks (72% of total cases) between June and November [10] and a lower rate from December to March [10–12]. Dengue is now considered to be regularly introduced in Vietnam from neighboring countries, i.e. Cambodia and Lao PDR, and recently, dengue was shown to initiate in Ho Chi Minh City and to expand north by successive waves [13].

Chikungunya virus (CHIKV), an alphavirus first isolated in Tanzania in 1953 [14, 15], has been detected since the 1960s in Asia and was reported in Vietnam in 1967 [15]. In 2004, a new variant of CHIKV emerged in East Africa and quickly spread over the Indian Ocean, India and Thailand, causing major outbreaks, [14–19] and was described in Cambodia in 2011 and 2012 and in Lao PDR [20]. This new variant had a high affinity for Aedes albopictus, which is actively transported via international trade maritime routes [16]. However, until now no clinical case of chikungunya has been described in Vietnam. Dengue and chikungunya symptoms are very similar, in particular in the early stages [21], making it easy to confuse diseases and underestimate the burden of chikungunya. In addition, the same Aedes mosquito species are vectoring both viruses and are widely prevalent in the region. Therefore, the present work was conducted in connection with the National Vietnamese Programme for surveillance of dengue to investigate the presence of dengue and chikungunya in patients hospitalized with acute fever and in mosquito populations present around the patient’s households as well as communes located in five provinces of North, Central and South Vietnam bordering Lao PDR and Cambodia. A specific focus was put on the identification of circulating mosquito haplotypes in these regions.

To our knowledge, this study is the first to investigate, in a comparative way, vector distribution, DENV and CHIKV circulation and occurrence of both inpatients hospitalized with acute febrile symptoms and mosquitoes across several provinces throughout Vietnam. The average rate of dengue among acute fever patients was about 3% and ranged between 2.06–5.70%, depending on the province. This is 10- to 5-times less than previously reported among acute fever inpatients [27, 28]. Hypotheses to explain this lower rate could be that this study focused on regional health centers, while suspected dengue cases might be directed to major hospitals not covered in this study. One cannot exclude that, since the previous studies in 2010 and 2013 used as reference were conducted [27, 28], the primary diagnostic for dengue has improved, leading to less undiagnosed dengue cases within acute fever inpatients. The presence of all four serotypes of dengue is in agreement with previous reports of the hyperendemicity status of Vietnam [8, 29].

Dengue is considered to be regularly introduced to Vietnam from Cambodia, mostly through human movement [13]. Furthermore, Raghwani et al. [13] demonstrated that infections initially started in Ho Chi Minh City and were moving north in successive waves. The higher incidence was observed in Ho Chi Minh City and urban areas, whereas a lower incidence was observed in rural areas. The low incidence reported in this work, which was conducted in rural areas, is in agreement with this previous report. This work brings a different and complementary insight by addressing, in addition to the rate of infection in acute fever inpatients, the rate of mosquito infection at, and in the vicinity of, the living places of infected patients. Under the hypothesis of a recurrent introduction of dengue from Cambodia, the Province of Long An, neighbor to Cambodia on one side and to Ho Chi Minh City on the other, was expected to display a higher rate of incidence. In fact, the incidence in human patients is one of the lowest here, despite a very high Breteau index, a proximity to high human population density and presence of the main cross border road system (Fig. 1). The province with the highest incidence was the southern province of Dac Nong. However, this province also displayed the lowest mosquito population density. The Breteau and Container indices here were the second lowest, whereas the House index was the highest, in agreement with the observed incidence rate. This indicates that, although widely used, the density of Aedes mosquitoes is not a reliable indicator of the risk of dengue. Although Ae. aegypti outnumbered Ae. albopictus, the latter appeared to represent a higher proportion of infected individuals. This is particularly true for the southern Province of Long An, where Ae. albopictus made up 50% of the infected mosquitoes while they only represented 1.2% of the mosquitoes sampled. In the northern/central provinces of Ha Tinh and Quang Tri, where Ae. albopictus made up 27–28% of the mosquitoes sampled, this species was the only one found to harbor dengue viruses. Interestingly, in Thua Thien Hue, where Ae. albopictus represented 47% of the captured mosquitoes, no infected individuals were found. These data emphasize the need to take these entomological indices with caution, as they can be misleading and do not reflect the risk of virus transmission. Furthermore, these indices are referring to the presence of species, whereas the ability of mosquitoes to efficiently transfer a virus is a trait linked to populations, i.e. subspecies [30–33]. Some specific populations, perhaps less numerous but more actively involved in the effective transmission of the virus, may have a higher impact. This would not be reflected by the entomological indices.

Another outcome of this work is the demonstration of the presence of multiple populations, i.e. maternally inherited mitochondrial cox1 lineages. All virus-infected mosquitoes described in this work, as well as most of the mosquitoes analyzed in this work, belonged to mitochondrial lineages, i.e. cox1 haplotypes, which have been described outside Southeast Asia. This feature was found in all provinces. Furthermore, a number of unknown Ae. aegypti cox1 haplotypes were sequenced, along with other species mistakenly field-identified as Ae. albopictus based on morphological traits. This simultaneous presence of several separate lineages in Vietnam indicates that there exists a high diversity of potential vectors. In addition, the description of the same haplotypes (populations) in other parts of the world indicates that a worldwide circulation of Aedes mosquito populations (or lineages) is taking place, and that these mosquito populations are transported worldwide including in Vietnam. Worldwide transportation and introduction of Aedes vectors is a wide spread phenomenon [30, 34, 35]. It is not possible to state whether these mosquito populations originated in Vietnam and were moved to other places where they were identified or, conversely, were moved to Vietnam from other parts of the world. Nevertheless, this worldwide description of the same haplotypes indicates a long distance transportation of these mosquitoes, and perhaps also of the viruses they carry, most likely due to international maritime trade [34].

This conclusion must be linked to elements previously reported. First, dengue virus was shown to undergo a complex dynamic involving both serotype and lineage replacements [8, 27, 28, 36]. Although many parameters could be involved in this mechanism, lineage replacement was thought to be more stochastic than selective [8, 36]. The worldwide circulation of mosquito populations, and the potential introduction of new virus lineages, could partly explain the regular virus replacement [8, 27, 28, 36] and the stochastic determinism of these replacements [8, 36]. Secondly, dengue virus has been shown in Vietnam to display a higher incidence in urban areas and, in particular, in Ho Chi Minh City which was described as the starting point of the north-bound expansion of dengue in Vietnam [13]. Ho Chi Minh City is located close to Cambodia, and the regular introduction of dengue virus from Cambodia to Ho Chi Minh City and then to the rest of Vietnam is very plausible. However, Ho Chi Minh City is also the economic capital of Vietnam and a main hub for the international transportation of goods. The network of ports in Ho Chi Minh City, known as Saigon Port, is the first one in size in Vietnam and 24th worldwide for containers. Introduction of mosquitoes through maritime international trade routes, in addition to introduction from Cambodia, is therefore also very plausible. Furthermore, no individual of the Cambodia 3 haplotype, described in Cambodia where the dengue virus is thought to have come from, was found to be infected with DENV or CHIKV. This work demonstrates that there is, therefore, no single local endemic population of mosquitoes involved in the transmission of dengue and chikungunya in Vietnam, but rather a countrywide co-circulation of several distinct populations of both Ae. aegypti and Ae. albopictus belonging to lineages distributed worldwide. Dengue-infected Ae. aegypti mosquitoes belonged to lineages described in Martinique, India and Thailand. With the exception of QT_LB_albo_55M1 which belonged to the haplotype alb9 of A. albopictus described in Thailand, all the other dengue-infected Ae. albopictus individuals belonged to the same population, which has no haplotype name and which was characterized in Romania and described as an invasive population in Europe [37]. The alb9 dengue-infected mosquito was a male, indicating the presence of vertical transmission, a trait already known for dengue [38–40].

One dengue-infected mosquito was found not to be Ae. albopictus but instead Culex vishnui. This is, to our knowledge, the first time C. vishnui, a species known for transmitting Japanese encephalitis, has been found infected with dengue. There is no evidence from the data reported here that C. vishnui can efficiently transmit dengue to humans through a blood meal. However, there is at least the demonstration that C. vishnui can take up the dengue virus and therefore be considered as a potential vector. Further research should be considered to investigate the occurrence of efficient transmission of dengue in this already medically-important mosquito group. It is also an indication that vector population studies must be extended to all mosquitoes species present, not Ae. aegypti and Ae. albopictus, when surveying the actual virus circulation in an ecosystem.

This work stresses the need to establish a genotype-based survey of circulating mosquitoes in Vietnam, not based on the species level as is currently done, but rather on the population at the infra-species level, with an extension to other local mosquito species. The entomological indices currently in use are misleading and other sets of indices reflecting the involvement of specific populations in the transmission must be developed. An integrative analysis encompassing the genetic study of viral lineages in human patients and in mosquitoes, along with the genotyping of mosquito populations, should be undertaken to provide clear information on the dynamics of dengue and chikungunya. Although no clinical chikungunya case was declared and no CHIKV was found in human febrile patients in this study, the detection of CHIKV in mosquito haplotypes bound to worldwide movements, to areas with major chikungunya outbreaks, indicate that the threat should be taken seriously and a dedicated surveillance program should be implemented. Dengue and chikungunya, and perhaps other Aedes-borne diseases, appear as global threats that should not be addressed at a national or even regional scale but rather at a global scale, with worldwide dimension characterized by permanent exchanges and movements.