Dengue fever is caused by the dengue virus (DENV), a single-stranded positive-sense RNA virus of the Flaviviridae family. It is the most recognized arbovirus in the world, transmitted by infected female mosquitoes, especially Aedes aegypti and Ae. albopictus (Hayes and Gubler, 1992). Dengue infections have increased exponentially in the majority of the tropics and subtropics over the past three decades. Currently, one third of the world’s population is susceptible to DENV infection. The WHO estimates that the global burden of dengue infection has increased about 10.3-fold in the last two decades (World Health Organization, 2023). This condition is primarily linked to uncontrolled urbanization, climate change, poor water supply and sewage management, rapid movement of people, animals, and trade via air and sea routes, and unsustainable vector control programmes (Wilder-Smith and Gubler, 2008; Gubler, 2011; Bhatia et al., 2022; Romanello et al., 2022).

Dengue infection is caused by four antigenically different serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 (Gubler, 2002) and the disease manifests itself in a range of ways, from asymptomatic infection and mild febrile illness (dengue fever) to more severe forms including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The most severe clinical manifestation, dengue shock syndrome (DSS), is characterized by coagulation abnormalities, hemorrhage, plasma leakage, and organ failure (Bhatt et al., 2021). The present dengue case classification, according to World Health Organization, comprises symptomatic patients with and without warning signals, as well as severe dengue (World Health Organization, 2009). Based on the genetic make-up of DENV each serotype has been subdivided into 4–5 genotypes (Weaver and Vasilakis, 2009). Furthermore, depending on their phylogenetic classification, each genotype has a range of lineages (Shrivastava et al., 2015). Infection with one serotype results in lifetime immunity against homologous serotypes, while there is no or limited immunity against infection to heterologous dengue serotypes. This secondary infection, which leads to enhanced dengue severity, is primarily caused by antibody-dependent enhancement (ADE), a mechanism mediated mostly by immunoglobulin G (IgG) (Teo et al., 2023). Additionally, certain lineages and genotypes have been associated with severe forms of dengue fever. These genetic variants with modest variations are also responsible for major outbreaks due to rapid transmission in both humans and mosquitoes (Messer et al., 2003). Hence, regular surveillance is necessary for the control and management of dengue.

Recent anthropogenic and climate-related changes have resulted in accelerated transmission of DENV, causing frequent dengue epidemics (Sarma et al., 2022). Previous studies suggested that several Southeast Asian countries demonstrated dengue hyperendemicity that was 18-fold higher than the Americas (Murray et al., 2013). Currently, almost all Indian states are under constant threat of dengue transmission. A nationwide dengue sero-survey during 2017–2018 showed that nearly half of the Indian population (48.7; 95% CI 43.5–54.0) was sero-positive for DENV (Murhekar et al., 2019). Since 2000, the north Indian states, including New Delhi, Rajasthan, Uttar Pradesh, Madhya Pradesh, Haryana, and Punjab, have reported dengue outbreaks at regular intervals (Mishra et al., 2015). Similarly, the western (Maharashtra), eastern (Odisha), and southern states (Kerala, Andhra Pradesh, and Telangana) also reported massive dengue outbreaks along with chikungunya (Anoop et al., 2010; Cecilia et al., 2011; Shrivastava et al., 2015). In view of this, there is an increasing demand for a safe and effective DENV vaccine that elicits immunity against all the four serotypes. At the moment, at least seven DENV vaccines are at various stages of clinical trials or pre-clinical investigations. Three of these, Dengvaxia^® (CYT-TDV), Qdenga^® (TAK-003) and TV003 (NIAD/Butantan/Merck) have shown promising results in clinical trials (Torres-Flores et al., 2022; Angelin et al., 2023). Dengvaxia^®, manufactured by Sanofi Pasteur, has been licensed in 20 countries and approved for use in individuals aged 6–45 years with laboratory confirmed previous dengue infection and living in endemic countries (Thomas and Yoon, 2019; Torres-Flores et al., 2022). However, shortcomings such as vaccine administration limited to dengue immune individuals only, non-availability in non-endemic countries and safety concerns in vaccine recipients who were dengue non-immune at the time of vaccine administration (Hadinegoro et al., 2015) in the case of Dengvaxia^® and less protection against DENV-3 (Rivera et al., 2022), as well as a lack of data for elderly individuals (Angelin et al., 2023) in the case of Qdenga^®, have posed significant challenges to the widespread and effective control of dengue fever through vaccination efforts. Until an effective vaccine becomes available, vector control will continue to be the primary approach for managing dengue transmission. Furthermore, systematic molecular monitoring of circulating DENV serotypes in mosquitoes and humans, as well as early detection of any serotype or genotype shift in a geographic area, would aid in the prediction of imminent DENV epidemics and devise targeted public health strategies for dengue control.

This study investigated the phylogenetic relationship of DENV-2 serotypes prevalent in central India (Bhopal, Madhya Pradesh) and reported the emergence of a new lineage (Genotype 4a, cosmopolitan non-Indian lineage) in naturally infected, wild-caught Ae. aegypti and Ae. albopictus mosquitoes. This study highlights the urgent need of DENV surveillance and clinical characterization of dengue fever in this strategically located part of the country.

A total of 2,371 adult female Aedes mosquitoes (1,498 Ae. aegypti and 873 Ae. albopictus) were collected. Of these, 1,890 mosquitoes (1,186 Ae. aegypti and 704 Ae. albopictus) were tested for DENV infection. Similarly, 370 Aedes larvae were tested for DENV infection in 37 pools (32 pools for Ae. aegypti and 5 pools for Ae. albopictus). Based on the RT-PCR amplification of C-PrM gene region (Supplementary Figure 1), DENV RNA was detected in seven Ae. aegypti and one Ae. albopictus individuals. Overall DENV infection rates were 0.59 and 0.14% in Ae. aegypti and Ae. albopictus, respectively. Area wise S_15 (Nizamuddin colony area) recorded the highest DENV infection rate (5.26%), while S_11 (Kotra Sultanabad area) recorded the lowest DENV infection rate (1.25%) for Ae. aegypti mosquitoes. Ae. albopictus was incriminated only in S_25 (Shakti Nagar) with an infection rate of 0.8% (Table 1). No DENV infection was detected in larval pools.

Phylogenetic analysis revealed that all eight DENV C-prM sequences were belonged to serotype 2 (Figure 2). Analysis of the representative serotype 2 C-prM sequences from India and other countries unambiguously clustered these 8 sequences with the non-Indian lineage of DENV-2 (Cosmopolitan genotype 4a) (Figures 3A, B). These sequences are substantially divergent from the other genotypes, with nucleotide divergence ranging from 7.4 to 22.2% (Supplementary Figure 2). The maximum clade credibility tree indicated a mean TMRCA of 7.4 years (95% HPD: 3.5–12.2) for the sequences in the present study, suggesting that this lineage was introduced in 2014 (95% HPD: 2009.8–2018.5) (Figure 4). Other isolates collected in Bhopal in 2016 (GenBank accession nos. MH051272-MH051275) grouped into two distinct clades within the most prevalent cosmopolitan 4b genotype, with identical TMRCA (2014, 95% HPD: 2011–2016), implying that both lineages of the DENV-2 cosmopolitan genotype circulated in Bhopal at the same time. When compared to DENV-2 reference strain KM204118 and other DENV-2 sequences, this lineage showed four significant amino acid alterations, namely E19A, M104I, L108M, and D143N (Supplementary Table 2). A statistically significant positive selection was observed in the DENV capsid protein at amino acid position 19 by different methods (MEME: p-value 0.05; FEL: p-value 0.09; and FUBER: probability 0.962).

Dengue is continuing to be the most serious arboviral infection in various Southeast and South Asian nations, including India. This is due to severe environmental changes, rapid urbanization, and increased human long-distance travel, which have resulted in the spread of Aedes species in previously unexplored regions (Hussain and Dhiman, 2022). The first virological dengue outbreak in India was reported in 1963 from Kolkata (East India) (Chatterjee et al., 1965). Both Ae. aegypti and Ae. albopictus have been identified as potential dengue vectors in different parts of the country, with varied incidences of all four known serotypes (Table 2). Central India, on the other hand, recorded its first dengue outbreak in 1966 in the Jabalpur district of Madhya Pradesh, with DENV-3 as the causative agent. Subsequently, other districts of Madhya Pradesh, such as Sagar (1966), Sarguja (1997), Gwalior (2002), and Bhopal and Indore (2009), also observed dengue outbreaks with DENV 1, 2, and 3 as causative agents (Sehgal et al., 1967; Mahadev et al., 1997; Parida et al., 2002; Kalpana et al., 2010). According to Mahadev et al. (1997), Ae. aegypti was the primary cause of the outbreaks in Madhya Pradesh.

All four serotypes of DENV have been linked to several outbreaks in India in the past (Guo et al., 2017), however, the majority of these outbreaks were associated with DENV-2 (Singh et al., 1999; Dash et al., 2006; Agarwal et al., 2023). The first DHF outbreak caused by DENV-2 occurred in New Delhi in 1996 (Dar et al., 1999). Since 2012, DENV infections in India have steadily increased, with active circulation of the DENV-2 serotype in different regions with varying prevalence (Sankari et al., 2012; Afreen et al., 2016; Murhekar et al., 2019; Alagarasu et al., 2021). However, the prevalence and distribution of DENV-2 in Aedes mosquitoes from Bhopal (Central India) remain unknown. The present study reports for the first time the circulation of DENV-2 serotype and its prevalence in both Ae. aegypti and Ae. albopictus collected from 29 different localities of Bhopal city. It has been observed that most of the DENV incriminated sites were in the South and South-western part of the city (Figure 1), which were also known to be the hot-spot area of dengue transmission in Bhopal city (Sarma et al., 2022). This finding further confirms the entomological risk for dengue transmission in these areas.

Dengue virus infection in wild-caught Aedes mosquitoes was assessed, and circulating DENV serotypes were characterized by sequence comparison and phylogenetic analysis in Bhopal city during the study. This is crucial because molecular characterization of these viruses aids in the identification of molecular subtypes or genotypes and the introduction of any new lineages. Previously, various regions of the dengue genome have been used for molecular phylogenetic analysis, but many studies have reported the C-prM gene as a tool in dengue virus genotyping (Murugesan et al., 2020; Titir et al., 2021). We retrieved previously reported C-prM gene sequences from the NCBI GenBank database for phylogenetic analysis.

It is evident that cosmopolitan genotypes of the DENV-2 virus are circulating across India (Table 1). However, when analyzing the phylogenetic relationship of DENV sequences generated in this study alongside other global sequences, it became evident that they clustered with non-Indian isolates, indicating the establishment of a novel lineage, lineage 4a, of DENV serotype 2 (Figure 3) in Aedes mosquitoes collected from Central India (Bhopal, Madhya Pradesh). Additionally, the MCC tree indicated that this lineage (4a) was introduced in Bhopal around 2013–2014. Previously, the introduction of a new genotype or lineage was proposed as the root cause of the transmission of a severe strain of dengue sickness in other parts of the world, such as the Americas (Rico-Hesse et al., 1997; Messer et al., 2003). It is pertinent to note that Bhopal experienced severe outbreaks of DENV in 2014 and 2016 (Agarwal et al., 2019), and since then the number of dengue cases has been increasing steadily in Bhopal (Sarma et al., 2022). This might be possible because lineages’ extinction and inevitable incursion were connected to a virus’s transmission bottleneck. For example, in Thailand, increased viral transmission by a mosquito vector was also recently connected to lineage replacement (Lambrechts et al., 2012). Similarly, Shrivastava et al. (2015) also reported the new lineage of DENV-3 (genotype 4) in eastern India, which was implicated in the dengue outbreak during the year 2011. However, in view of the scarcity of molecular data on the circulating serotypes/genotypes during that time, pinpointing the lineage that possibly triggered those outbreaks is difficult. The MCC tree shows both the lineages (4a and 4b of the cosmopolitan genotype) were circulating in Bhopal, perhaps with different degrees of dominance. The same has been evidenced by a recent hospital based study in the same area, where out of total 154 RT-PCR positive dengue cases, majority (66 nos.) of them belonged to DENV-2 serotype and of these 66 DENV isolates, 13 were sequenced of which 12 were found to belonged to genotype 4a during 2019 and 2021. It was also observed that the new lineage of DENV-2 was also associated with a longer duration of hospitalization (>10 ± 3 days) and hemorrhagic manifestations indicating its role in severity if disease in the studied area (Agarwal et al., 2023). Higher evolutionary and increased transmission rates of one DENV genotype may outnumber the others in the community, resulting in a lineage shift. This shift may be due to several factors, including changes in the mosquito vector population, host immunity, environmental conditions, and international travel (Shrivastava et al., 2015). Bhopal, a central Indian city, is a favorite destination for international tourists and received 316,195 international travelers during 2014 (India Tourism Statistics, 2014). Although most of the incident dengue cases are of local origin, there are some immigrated cases also, which, along with the international travelers, might facilitates the introduction of this new lineage to the study area.

Aedes mosquitoes have the ability to transmit the virus to their offspring, which is identified as vertical transmission. This phenomenon is important to understand as it may contribute to the existence of viruses in the environment in the absence of susceptible hosts. This phenomenon may also be linked to disease endemism, implying that vertical transmission plays an important role in the establishment of endemicity (Hull et al., 1984). The presence and significance of this transmission in Aedes have been reported in previous studies from different regions of the world (Martins et al., 2012; Buckner et al., 2013; Wijesinghe et al., 2021). Within India, natural vertical transmission of DENV was observed in dengue hyper-endemic areas such as Rajasthan (Angel and Joshi, 2008; Bina et al., 2008), Tamil Nadu (Arunachalam et al., 2008), New Delhi (Bina et al., 2008), and Kerala (Thenmozhi et al., 2007). Although no DENV infection was observed in the larvae of Ae. aegypti and Ae. albopictus in the present study, which may be due to low to mid endemicity of the disease in the studied area and low number of larvae processed, the presence of natural vertical transmission of DENV in the studied area couldn’t be ruled out. More molecular surveillance with large numbers larvae is required to confirm the natural vertical transmission of DENV in field collected Aedes mosquitoes in this part of India.

The present study, for the first time, confirms the prevalence of DENV serotype 2 (genotype 4) in field collected Aedes mosquitoes from Bhopal city, Central India. The phylogenetic placement of the DENV sequences with non-Indian lineages indicates the circulation of a new lineage (4a) of cosmopolitan genotype. This lineage transition of DENV-2 is a major cause of concern and requires routine surveillance of viral circulation throughout endemic and non-endemic areas for a better understanding of transmission dynamics and effective control and management of the dengue burden in this central part of India.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

DS: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing—original draft. LR: Data curation, Formal Analysis, Investigation, Methodology, Writing—review and editing. SM: Data curation, Formal Analysis, Investigation, Methodology, Writing—review and editing. DD: Investigation, Methodology, Visualization, Writing—review and editing. AA: Formal Analysis, Investigation, Methodology, Visualization, Writing—review and editing. GS: Formal Analysis, Methodology, Writing—original draft. TS: Formal Analysis, Methodology, Writing—review and editing. MKw: Visualization, Writing—review and editing. SaS: Investigation, Writing—review and editing. VV: Visualization, Writing—review and editing. MK: Investigation, Methodology, Writing—review and editing. SwS: Visualization, Writing—review and editing. RT: Supervision, Writing—review and editing. AP: Conceptualization, Methodology, Supervision, Writing—review and editing.