Co-infection of dengue and Zika viruses mutually enhances viral replication in the mosquito Aedes aegypti

Lin, Daniel Chieh-Ding; Weng, Shih-Che; Tsao, Po-Nien; Chu, Justin Jang Hann; Shiao, Shin-Hong

doi:10.1186/s13071-023-05778-1

Co-infection of dengue and Zika viruses mutually enhances viral replication in the mosquito Aedes aegypti

Research
Open access
Published: 11 May 2023

Volume 16, article number 160, (2023)
Cite this article

Download PDF

You have full access to this open access article

Parasites & Vectors Aims and scope Submit manuscript

Co-infection of dengue and Zika viruses mutually enhances viral replication in the mosquito Aedes aegypti

Download PDF

Daniel Chieh-Ding Lin¹^na1,
Shih-Che Weng²^na1,
Po-Nien Tsao^3,4,
Justin Jang Hann Chu⁵ &
…
Shin-Hong Shiao²

4162 Accesses
5 Citations
18 Altmetric
Explore all metrics

Abstract

Background

The mosquito Aedes aegypti transmits two of the most serious mosquito-borne viruses, dengue virus (DENV) and Zika virus (ZIKV), which results in significant human morbidity and mortality worldwide. The quickly shifting landscapes of DENV and ZIKV endemicity worldwide raise concerns that their co-circulation through the Ae. aegypti mosquito vector could greatly exacerbate the disease burden in humans. Recent reports have indicated an increase in the number of co-infection cases in expanding co-endemic regions; however, the impact of co-infection on viral infection and the detailed molecular mechanisms remain to be defined.

Methods

C6/36 (Aedes albopictus) cells were cultured in Dulbecco's modified Eagle medium/Mitsuhashi and Maramorosch Insect Medium (DMEM/MM) (1:1) containing 2% heat-inactivated fetal bovine serum and 1× penicillin/streptomycin solution. For virus propagation, the cells were infected with either DENV serotype 2 (DENV2) strain 16681 or ZIKV isolate Thailand/1610acTw (MF692778.1). Mosquitoes (Ae. aegypti UGAL [University of Georgia Laboratory]/Rockefeller strain) were orally infected with DENV2 and ZIKV through infectious blood-feeding.

Results

We first examined viral replication activity in cells infected simultaneously, or sequentially, with DENV and ZIKV, and found interspecies binding of viral genomic transcripts to the non-structural protein 5 (NS5). When we challenged Ae. aegypti mosquitos with both DENV2 and ZIKV sequentially to probe similar interactions, virus production and vector susceptibility to infection were significantly enhanced.

Conclusions

Our results suggest that DENV2 and ZIKV simultaneously establishing infection in the Ae. aegypti mosquito vector may augment one another during replication. The data also implicate the homologous NS5 protein as a key intersection between the flaviviruses in co-infection, highlighting it as a potential target for vector control.

Sindbis virus interferes with dengue 4 virus replication and its potential transmission by Aedes albopictus

Article Open access 30 January 2015

Mosquito densovirus significantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes (Diptera: Culicidae)

Article Open access 09 May 2023

Mosquito vector competence for dengue is modulated by insect-specific viruses

Article 05 January 2023

Background

Mosquito-borne diseases are one of the most significant public health burdens [1,2,3,4,5]. Human activities, urbanization, and climate change are increasingly bringing more human hosts in contact with disease vectors [2, 6,7,8,9]. Currently, half of the global population is at risk for dengue virus (DENV) infection [2, 8, 10]. In the aftermath of the 2015–2016 Zika virus (ZIKV) outbreak, which exposed more than 130 million people to infection, the virus remains endemic to tropical and subtropical regions [11].

Sharing a common vector in Aedes aegypti, DENV and ZIKV endemicity may expand in concert, resulting in widespread co-circulation [12, 13]. Thus, DENV-ZIKV synergy presents a bleak outlook for the near future with a growing proportion of the global population living under threat of simultaneous infection by both flaviviruses. As DENV-ZIKV co-circulation expands worldwide to affect currently low-risk or virus-free regions, mosquito vectors will have increased opportunities to receive and transmit both viruses [5]. Recent reports of DENV-ZIKV co-infection corroborate this prediction and reflect a proliferating synergy that has been overlooked. This may be the result of systemic underreporting engendered by difficulties in the differential diagnosis and detection of asymptomatic infections [14,15,16].

One cross-sectional study of the ZIKV epidemic in Colombia detected 8.8% of the DENV-ZIKV serotype among 34 co-infection cases [15]. Another study in southern Mexico randomly sampled a cohort of pregnant women during a non-epidemic period and found a relatively high proportion (2%) with DENV-ZIKV co-infection [17]. A previous report revealed that DENV infections occurred during the same period, highlighting the concerning extent of silent transmission of ZIKV with DENV [17, 18]. Besides underreporting, underlying this phenomenon may be antibody-dependent enhancement (ADE) between DENV and ZIKV in co-endemic areas. Studies support ADE of ZIKV infection by anti-DENV antibodies [4, 19,20,21], which not only may increase disease severity, but also may drive ZIKV transmission into primarily DENV-endemic areas. Similar results have been suggested for DENV, in which ZIKV-mediated ADE increases the propensity for severe disease and enables DENV to persist and proliferate in primarily ZIKV-endemic regions [

Co-infection of Ae. aegypti with DENV2 and ZIKV results in differential viral genome expression

Finding significant enhancement of viral replication by DENV2-ZIKV co-infection in vitro possibly arising from cross-species interactions, we challenged adult female Ae. aegypti sequentially with both viruses to quantify the effects of DENV2-ZIKV co-infection on vector competence in vivo. So far, existing studies into DENV-ZIKV co-infection have only challenged Ae. aegypti simultaneously, presenting both viruses in the same blood meal (BM). Contrarily, we elected to challenge mosquitos with DENV2 and ZIKV sequentially to maximize ecological validity. It is much more likely that in a DENV-ZIKV co-endemic area, a female mosquito would acquire co-infection in sequential feeding episodes: by feeding first on a DENV-infected host, then a ZIKV-infected host, or vice versa. The possibility of a mosquito obtaining both viruses from a DENV-ZIKV co-viremic human host through a single BM is much lower. Critically, we also consider the findings of studies reporting that non-infectious BM prior to subsequent viral BM can promote viral replication, as the initial non-infectious BM induces physiological changes in the midgut epithelium, rendering it more permissible to dissemination through the basal lamina [28, 29, 33, 34].

Thus, we included cohorts of mosquitoes presented with an initial naïve BM before challenge with DENV2 or ZIKV to account for this possible confounder in evaluating co-infection effects on viral replication (Fig. 4A). Overall, two co-infection schemes were used in vivo, in which Ae. aegypti were either challenged first with DENV2 and then ZIKV on a second BM (DENV2 → ZIKV), or vice versa (ZIKV → DENV2). These cohorts were compared against mosquitos single-infected with DENV and ZIKV, through either one or two BMs, as well as a mock cohort (BM-BM).

Collecting mosquitos at 7 dpi, which we previously determined to be an optimal time point for viral genomic analysis, we quantified via qRT-PCR the relative expression of DENV2 and ZIKV genomes, respectively (Fig. 4A). We found that, contrary to our observations in vitro, DENV2 expression was significantly downregulated in both co-infection scenarios, particularly when compared with those challenged twice (DENV2 → DENV2) with DENV2 (Fig. 4B). Also, a significant difference in expression between DENV2 single-infected mosquitos presented with (BM → DENV2), and without (sugar-fed → DENV2), an initial naïve BM suggests that BM-induced modifications do indeed promote viral replication by encouraging dissemination from the midgut. Genomic expression of ZIKV was significantly elevated in both co-infection scenarios, even in comparison to mosquitos infected twice (ZIKV → ZIKV) with ZIKV (Fig. 4C). Between the two co-infection groups, ZIKV → DENV2 mosquitos expressed genomic ZIKV at significantly higher levels than did their DENV2 → ZIKV counterparts. There was also a significant difference in expression between sugar-fed → ZIKV and BM → ZIKV individuals, as with DENV2, again highlighting the initial BM’s ability to promote viral replication in and past the midgut. Moving forward, we evaluated the implications of co-infection on vector competence, quantifying the infectivity of virus particles produced in vivo.

Virus production and vector susceptibility are enhanced in Ae. aegypti challenged with both DENV2 and ZIKV

Having characterized the genomic expression profiles of co-infected mosquitos, we next assessed the quantity and infectivity of viruses produced therein. Performing plaque assays on isolated virus from individuals collected at 7 dpi from each treatment group (Fig. 4A), we found that viral titers were significantly higher in co-infected mosquitos than in the DENV single-infected cohort (Fig. 5A). Notably, these increases were observed for both DENV2 → ZIKV and ZIKV → DENV2 co-infection groups against all three DENV single-infection cohorts: those challenged either once (sugar/BM → DENV2) or twice (DENV2 → DENV2) with DENV2. Accordingly, the corresponding infection rates were markedly higher in co-infected mosquitos, increasing by as much as 36.7% (Fig. 5B). Compared with ZIKV single-infected cohorts, viral titers were also higher in co-infected individuals. Infection rates also saw increases by about 10% except when compared with the sugar → ZIKV cohort, a result likely attributable to individual variability. Despite the unexpectedly high infection rate, the median viral titer for this cohort was at least twofold lower than those of co-infected cohorts. Overall, mosquitos challenged sequentially with DENV and ZIKV produced more infectious virus, which induced a greater extent of infection ex vivo.

Mosquitos twice-challenged with ZIKV produced higher viral titers than those co-infected, an unsurprising result following consecutive ZIKV infection. Secondary infection is greatly assisted by the existing RC infrastructure and an infected, compromised midgut epithelium and basal lamina (ZIKV → ZIKV). Indeed, viral titers were higher in co-infected mosquitos compared with those challenged once (sugar/BM → ZIKV). Infection rates of co-infected mosquitos also increased, suggesting greater susceptibility to infection and virus propagation, i.e., enhanced vector competence. As for mosquitos twice-challenged with DENV2, the clear enhancement of viral replication by co-infection is much more readily apparent, as these cohorts were entirely refractory to infection (0% infected, DENV2 → DENV2). Across DENV2 single-infected cohorts, infection rates peaked at 11.1%. The fact that vector infection rates were elevated to 44.1% and 46.7%, respectively, in co-infected cohorts indicates that DENV2-ZIKV co-infection positively, mutually modulates viral replication.

Of note, mosquitos given an initial naïve BM neither produced significantly higher viral titers nor were infected at higher rates than those directly challenged with DENV2/ZIKV. This suggests that while an initial non-infectious BM may correlate with higher viral genomic expression by assisting virus dissemination from the midgut, it ultimately neither enhances the production of viable, infectious particles nor promotes co-infected Ae. aegypti susceptibility to infection.

Taken together, our results show that DENV2-ZIKV co-infection significantly enhances virus production and vector susceptibility to infection. At the cellular level, viral replication is mutually enhanced owing to the overlap of highly homologous flaviviral replication machinery. We observed therein that DENV2 transcripts engage ZIKV-NS5. Overall, our findings raise grave concerns about DENV2 and ZIKV co-circulation, which threatens to strain healthcare resources and exacerbate transmission and disease as mosquitos vector these flaviviruses with greater efficiency.

Discussion

DENV and ZIKV are flaviviruses transmitted by mosquitos of the Aedes genus, primarily Ae. aegypti [35]. They co-circulate in overlap** endemic areas. Consequently, as the spread of DENV and ZIKV expands rapidly, mosquitos will have increased opportunities to acquire simultaneous and/or mixed infections with different types of flaviviruses [35]. This may occur following an infectious BM from a single human co-viremic for DENV and ZIKV, or when mosquitos acquire sequential BMs from two individuals, each infected with a different virus. DENV and ZIKV share a highly conserved non-structural protein repository consisting of five enzymes/subunits (NS1–5), which associate closely to form a tightly-regulated RC. We hypothesized that DENV and ZIKV interact through their homologous RC components during replication, which has significant implications for viral replication and vector competence.

In the present study, we observed significant enhancements in virus production and vector susceptibility following DENV2-ZIKV co-infection in Ae. aegypti, and we demonstrated the cellular and molecular bases of these effects. In co-infected mosquito cells, we found that DENV2 expression was significantly enhanced, whereas ZIKV was coincidentally markedly suppressed and virus production was significantly increased for both. The finding that flaviviral RCs co-localize extensively in the cytoplasm, we determined whether DENV and ZIKV participate in cross-species interactions during replication. We found that the replicating DENV2 genome engages ZIKV-NS5. This surprising interaction readily explains the drastic enhancement of DENV2 expression in vitro, which suggests that DENV2 engages the ZIKV RC competitively. DENV-ZIKV interactions during replication provide a basis for the mutual increase in secretory virus particles observed. Next, we challenged Ae. aegypti adult females with both viruses to determine whether similar DENV2-ZIKV interactions also modulate viral replication in vivo. We found that while genomic expression at 7 dpi of DENV2 was markedly downregulated, with ZIKV upregulated, there was again a mutual enhancement of viral replication as indicated by significantly elevated levels of infectious virions produced in co-infected mosquitos, which were also more susceptible to infection.

Our findings describe for the first time the potent mutualistic outcomes of flaviviral co-infection for viral replication, and suggest that vector competence may be enhanced as a result. Vector competence in the arboviral sylvatic cycle is defined by the extent to which the mosquito permits a virus to utilize its circulatory system. The virus must first establish infection in the midgut and produce virions that disseminate to secondary tissues, which must then sustain replication throughout the organ system until freshly propagated virions breach the salivary glands. We found that viruses produced in DENV-ZIKV co-challenged mosquitos were significantly more abundant compared with those infected with only one virus, particularly DENV2. Moreover, these mosquitos were infected at a higher rate, suggesting that co-infected mosquitos are more susceptible to infection, perhaps resulting from the convergence of immune suppression pathways of both replicating viruses, particularly in the midgut. These results strongly suggest that co-infection enhances vector competence because, at a collection date of 7 dpi, either virus will have already disseminated from the midgut and commenced replication throughout the entire body to produce viable, infectious particles. Although employing more collection points to visualize replication kinetics may allow for a clearer spatio-temporal resolution, for the purpose of understanding co-infection in terms of viral replication and vector competence, it was sufficient to isolate virus from mosquitos at 7 dpi for the plaque assay. The implications of our results for vector competence are somewhat limited, however, absent organ-specific analysis. Specifically, quantifying virus present in the salivary glands may permit more direct observation of outcomes for vector competence, as infectious viral particles must be secreted into the saliva during a BM for transmission [56]. However, the variability of flaviviral infection in vivo among individual mosquitos largely precluded such an investigation, as salivary glands dissected and analyzed individually would likely vary greatly in viral titer. Collecting whole bodies enabled an individual analysis without sacrificing statistical integrity. Additionally, we studied replication dynamics in vivo using a blood meal challenge, opting not to infect mosquitos by intrathoracic injection to preserve the critical barrier to vector competence manifest in the midgut’s physical and immunological fortifications. Quantifying virus propagated through sequential co-infection by oral challenge allowed us to observe the consequences of DENV-ZIKV interaction directly through the entire course of infection within the vector, beginning with ingestion in the midgut. Furthermore, our findings are highly relevant to the evolving landscape of DENV-ZIKV endemicity, as our sequential oral infection model is closely aligned with actual vector activity. Mosquitos are much more likely to acquire flaviviral co-infection sequentially from individual hosts than from a single host viremic for both DENV and ZIKV. Individual variability in feeding opportunity, i.e., extent of engorgement, however, likely negates many real differences in virus intake between laboratory and field.

To date, exploratory studies into arboviral co-infection have only established that Ae. aegypti are susceptible to infection by more than one type of virus and they may simultaneously transmit multiple viruses. One study reported that Ae. aegypti simultaneously challenged with a combination of two or three arboviruses including DENV2, ZIKV, and chikungunya (CHIKV), were frequently double- or triple-infected, which indicated that the mosquitos are susceptible to co-infection. Inoculation of saliva in vitro confirmed the potential for co-transmission of all three viruses [12]. Another study found that mosquitos simultaneously co-infected with DENV and ZIKV preferably transmit the latter [24]. The co-infection and co-transmission potential of ZIKV-CHIKV [36, 37] and DENV-CHIKV [38] have also been supported. With respect to arboviral co-infection, our study contributes novel insight into its implications for viral propagation in Ae. Aegypti and demonstrates that DENV-ZIKV co-infection mutually enhances viral replication. Our results also suggest that vector competence may be enhanced following DENV-ZIKV co-infection, as indicated by increased infection rates. We also provide the first account of molecular mechanisms underlying co-infection effects by reporting that DENV engages ZIKV-NS5 during replication.

As for the apparent conflict between our in vitro and in vivo results in which DENV2 replication appeared be competitively promoted in co-infected cells (Figs. 1B, 2C), but drastically suppressed in the mosquito (Fig. 4), it is important to note that markedly reduced DENV2 expression in co-infected mosquitos reflected only the viral genome content at 7 dpi, not the amount of infectious DENV virions produced by the vector. Indeed, virus production was significantly higher in co-infected mosquitos than in those single-infected with DENV. In addition, co-infected cohorts were much more susceptible to infection. Low DENV2 genomic expression suggests that there may be competitive engagement between ZIKV and DENV2 due to interaction between ZIKV genomic transcripts with DENV2 RC proteins. We showed that DENV2 utilizes ZIKV-NS5 for transcription; thus, it is likely that ZIKV may reciprocally utilize DENV2-NS5 to its advantage. Another intriguing possibility is that flaviviral NS5 may have a dual purpose in the convergence of DENV-ZIKV co-infection by cross-species cap** of the respective RNA genomes by N-terminus methyltransferases to assist in evasion of the host immune response. This is an alternative (and not mutually exclusive) molecular premise for the observed increases in virus production and vector susceptibility in co-infected mosquitos. Further biochemical studies regarding the DENV-ZIKV interaction via NS5, a possible target for vector control and vaccine development, is warranted as the co-circulation of DENV and ZIKV broadens globally.

The clinical and epidemiological implications of expanding flaviviral co-circulation remain largely unexplored. Though it is not clear whether co-infected patients develop more severe disease [39,40,41,42], increased co-circulation and transmission of multiple flaviviruses will surely pose significant problems for diagnosis and surveillance because of the common clinical presentation, asymptomatic response, and cross-reactivity. Although progress is being made on the diagnostics front [43,44,45], effective vector control remains the most effective approach to managing and eliminating mosquito-borne diseases [3]. Central to vector control is a clear understanding of the pathogen-vector relationship as it evolves in real-time, with expanding arboviral co-circulation being a worrying trend that we have addressed. As the periphery of DENV-ZIKV co-circulation expands, it also encompasses other arboviruses, such as CHIKV [46]. As we demonstrated, co-infection with DENV and ZIKV mutually enhances viral replication within the vector. Further interaction with other arbovirus, such as yellow fever virus or CHIKV, may result in unknown synergistic effects. This may similarly threaten to facilitate widespread circulation and transmission of multiple deadly viruses by modulating the vector response. The interplay among these arboviruses in Ae. aegypti and its effects on viral replication and vector competence require further study. Future work in this area could incorporate the study of viral interactions with the mosquito microbiota [47, 48] and diverse RNA responses [49,50,51,52] of the mosquito. Whether differential interactions arise between the DENV serotypes in co-infection scenarios is also worth pursuing, as all four DENV serotypes (DENV1–4) are spreading throughout Asia, Africa, and the Americas [53]. It also remains to be determined whether arbovirus co-infection influences virus selection pressure and recombination events [54, 55]. In conclusion, this study presents the novel finding that DENV-ZIKV co-infection mutually enhances viral replication within the mosquito. This threatens to increase the disease burden in co-endemic areas, drive the “silent” transmission of strains not predominantly circulating, and introduce flaviviruses into communities not yet seen. With arboviral co-endemicity on the rise globally, the rapidly shifting vector–pathogen relationship must be further investigated, in which the pathogen itself bears many faces.

Conclusions

In this study, we determined the effects of DENV and ZIKV co-infection on viral replication in Ae. aegypti and to identify the specific molecular interactions involved. We first examined viral replication dynamics in cells infected simultaneously or sequentially with DENV and ZIKV. We report the interspecies binding of viral genomic transcripts to NS5. We then challenged Ae. aegypti mosquitos with both DENV2 and ZIKV sequentially to identify similar interactions, and found that virus production and vector susceptibility to infection were significantly enhanced. Our results suggest that DENV2 and ZIKV simultaneously establish infection in the Ae. aegypti vector, which may mutually augment one another during replication. The data also implicate the homologous NS5 protein as a key intersection between the flaviviruses in co-infection, highlighting it as a potential target for vector control.

Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

BM:: Blood meal
BSA:: Bovine serum albumin
dpi:: Days post-infection
DENV:: Dengue virus
ICR:: Institute of Cancer Research
IFA:: Immunofluorescence assay
MOI:: Multiplicity of infection
PE:: Post-eclosion
RCs:: Replication complexes
ZIKV:: Zika virus

References

Wilder-Smith A, Ooi EE, Horstick O, Wills B. Dengue. Lancet. 2019;393:350–63. https://doi.org/10.1016/S0140-6736(18)32560-1.
Article PubMed Google Scholar
Wellekens K, Betrains A, De Munter P, Peetermans W. Dengue: current state one year before WHO 2010–2020 goals. Acta Clin Belg. 2020. https://doi.org/10.1080/17843286.2020.1837576.
Article PubMed Google Scholar
Wilson AL, Courtenay O, Kelly-Hope LA, Scott TW, Takken W, Torr SJ, et al. The importance of vector control for the control and elimination of vector-borne diseases. PLoS Negl Trop Dis. 2020;14:e0007831. https://doi.org/10.1371/journal.pntd.0007831.
Article CAS PubMed PubMed Central Google Scholar
Wang WH, Urbina AN, Wu CC, Lin CY, Thitithanyanont A, Assavalapsakul W, et al. An epidemiological survey of the current status of Zika and the immune interaction between dengue and Zika infection in Southern Taiwan. Int J Infect Dis. 2020;93:151–9. https://doi.org/10.1016/j.ijid.2020.01.031.
Article CAS PubMed Google Scholar
Achee NL, Grieco JP, Vatandoost H, Seixas G, Pinto J, Ching-Ng L, et al. Alternative strategies for mosquito-borne arbovirus control. PLoS Negl Trop Dis. 2019;13:e0006822. https://doi.org/10.1371/journal.pntd.0006822.
Article CAS PubMed PubMed Central Google Scholar
Shepard DS, Undurraga EA, Halasa YA, Stanaway JD. The global economic burden of dengue: a systematic analysis. Lancet Infect Dis. 2016;16:935–41. https://doi.org/10.1016/S1473-3099(16)00146-8.
Article PubMed Google Scholar
Hiscott J, Wilder-Smith A. Editorial overview: the challenge to defeat dengue. Curr Opin Virol. 2020;43:iii–v. https://doi.org/10.1016/j.coviro.2020.10.004.
Article PubMed Google Scholar
Cattarino L, Rodriguez-Barraquer I, Imai N, Cummings DAT, Ferguson NM. Map** global variation in dengue transmission intensity. Sci Transl Med. 2020;12:528. https://doi.org/10.1126/scitranslmed.aax4144.
Article Google Scholar
Stanaway JD, Shepard DS, Undurraga EA, Halasa YA, Coffeng LE, Brady OJ, et al. The global burden of dengue: an analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis. 2016;16:712–23. https://doi.org/10.1016/S1473-3099(16)00026-8.
Article PubMed PubMed Central Google Scholar
Brady OJ, Gething PW, Bhatt S, Messina JP, Brownstein JS, Hoen AG, et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis. 2012;6:e1760. https://doi.org/10.1371/journal.pntd.0001760.
Article PubMed PubMed Central Google Scholar
Attaway DF, Waters NM, Geraghty EM, Jacobsen KH. Zika virus: Endemic and epidemic ranges of Aedes mosquito transmission. J Infect Public Health. 2017;10:120–3. https://doi.org/10.1016/j.jiph.2016.09.008.
Article PubMed Google Scholar
Ruckert C, Weger-Lucarelli J, Garcia-Luna SM, Young MC, Byas AD, Murrieta RA, et al. Impact of simultaneous exposure to arboviruses on infection and transmission by Aedes aegypti mosquitoes. Nat Commun. 2017;8:15412. https://doi.org/10.1038/ncomms15412.
Article CAS PubMed PubMed Central Google Scholar
Sukhralia S, Verma M, Gopirajan S, Dhanaraj PS, Lal R, Mehla N, et al. From dengue to Zika: the wide spread of mosquito-borne arboviruses. Eur J Clin Microbiol Infect Dis. 2019;38:3–14. https://doi.org/10.1007/s10096-018-3375-7.
Article PubMed Google Scholar
Pessoa R, Patriota JV, Lourdes de Souza M, Felix AC, Mamede N, Sanabani SS. Investigation into an outbreak of dengue-like illness in Pernambuco, Brazil, revealed a Cocirculation of Zika, Chikungunya, and Dengue virus type 1. Medicine (Baltimore). 2016;95:e3201. https://doi.org/10.1097/MD.0000000000003201.
Article PubMed Google Scholar
Mercado-Reyes M, Acosta-Reyes J, Navarro-Lechuga E, Corchuelo S, Rico A, Parra E, et al. Dengue, chikungunya and zika virus coinfection: results of the national surveillance during the zika epidemic in Colombia. Epidemiol Infect. 2019;147:e77. https://doi.org/10.1017/S095026881800359X.
Article PubMed PubMed Central Google Scholar
Estofolete CF, Terzian ACB, Colombo TE, de Freitas Guimaraes G, Ferraz HCJ, da Silva RA, et al. Co-infection between Zika and different Dengue serotypes during DENV outbreak in Brazil. J Infect Public Health. 2019;12:178–81. https://doi.org/10.1016/j.jiph.2018.09.007.
Article PubMed Google Scholar
Eligio-Garcia L, Crisostomo-Vazquez MDP, Caballero-Garcia ML, Soria-Guerrero M, Mendez-Galvan JF, Lopez-Cancino SA, et al. Co-infection of Dengue, Zika and Chikungunya in a group of pregnant women from Tuxtla Gutierrez, Chiapas: preliminary data 2019. PLoS Negl Trop Dis. 2020;14:e0008880. https://doi.org/10.1371/journal.pntd.0008880.
Article CAS PubMed PubMed Central Google Scholar
Sharp TM, Fischer M, Munoz-Jordan JL, Paz-Bailey G, Staples JE, Gregory CJ, et al. Dengue and Zika virus diagnostic testing for patients with a clinically compatible illness and risk for infection with both viruses. MMWR Recomm Rep. 2019;68:1–10. https://doi.org/10.15585/mmwr.rr6801a1.
Article PubMed PubMed Central Google Scholar
Paul LM, Carlin ER, Jenkins MM, Tan AL, Barcellona CM, Nicholson CO, et al. Dengue virus antibodies enhance Zika virus infection. Clin Transl Immunol. 2016;5:e117. https://doi.org/10.1038/cti.2016.72.
Article CAS Google Scholar
Castanha PMS, Nascimento EJM, Braga C, Cordeiro MT, de Carvalho OV, de Mendonca LR, et al. Dengue virus-specific antibodies enhance Brazilian Zika virus infection. J Infect Dis. 2017;215:781–5. https://doi.org/10.1093/infdis/jiw638.
Article CAS PubMed Google Scholar
Olawoyin O, Kribs C. Coinfection, altered vector infectivity, and antibody-dependent enhancement: the Dengue-Zika interplay. Bull Math Biol. 2020;82:13. https://doi.org/10.1007/s11538-019-00681-2.
Article CAS PubMed PubMed Central Google Scholar
Tang B, **ao Y, Sander B, Kulkarni MA, Radam-Lac Research T, Wu J. Modelling the impact of antibody-dependent enhancement on disease severity of Zika virus and dengue virus sequential and co-infection. R Soc Open Sci. 2020;7:191749. https://doi.org/10.1098/rsos.191749.
Article CAS PubMed PubMed Central Google Scholar
Katzelnick LC, Narvaez C, Arguello S, Lopez Mercado B, Collado D, Ampie O, et al. Zika virus infection enhances future risk of severe dengue disease. Science. 2020;369:1123–8. https://doi.org/10.1126/science.abb6143.
Article CAS PubMed PubMed Central Google Scholar
Chaves BA, Orfano AS, Nogueira PM, Rodrigues NB, Campolina TB, Nacif-Pimenta R, et al. Coinfection with Zika virus (ZIKV) and Dengue virus results in preferential ZIKV transmission by vector bite to vertebrate host. J Infect Dis. 2018;218:563–71. https://doi.org/10.1093/infdis/jiy196.
Article PubMed PubMed Central Google Scholar
Weng SC, Shiao SH. Frizzled 2 is a key component in the regulation of TOR signaling-mediated egg production in the mosquito Aedes aegypti. Insect Biochem Mol Biol. 2015;61:17–24. https://doi.org/10.1016/j.ibmb.2015.03.010.
Article CAS PubMed Google Scholar
Chang CH, Liu YT, Weng SC, Chen IY, Tsao PN, Shiao SH. The non-canonical Notch signaling is essential for the control of fertility in Aedes aegypti. PLoS Negl Trop Dis. 2018;12:e0006307. https://doi.org/10.1371/journal.pntd.0006307.
Article CAS PubMed PubMed Central Google Scholar
Sri-In C, Weng SC, Chen WY, Wu-Hsieh BA, Tu WC, Shiao SH. A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission. Insect Biochem Mol Biol. 2019;111:103181. https://doi.org/10.1016/j.ibmb.2019.103181.
Article CAS PubMed Google Scholar
Chen TH, Lo YP, Yang CF, Chen WJ. Additive protection by antioxidant and apoptosis-inhibiting effects on mosquito cells with dengue 2 virus infection. PLoS Negl Trop Dis. 2012;6:e1613. https://doi.org/10.1371/journal.pntd.0001613.
Article CAS PubMed PubMed Central Google Scholar
Chen TH, Tang P, Yang CF, Kao LH, Lo YP, Chuang CK, et al. Antioxidant defense is one of the mechanisms by which mosquito cells survive dengue 2 viral infection. Virology. 2011;410:410–7. https://doi.org/10.1016/j.virol.2010.12.013.
Article CAS PubMed Google Scholar
Weng SC, Zhou YX, Shiao SH. A flavivirus-inducible gene expression system that modulates broad-spectrum antiviral activity against dengue and Zika viruses. Insect Biochem Mol Biol. 2022;142:103723. https://doi.org/10.1016/j.ibmb.2022.103723.
Article CAS PubMed Google Scholar
Weng SC, Tsao PN, Shiao SH. Blood glucose promotes dengue virus infection in the mosquito Aedes aegypti. Parasit Vectors. 2021;14:376. https://doi.org/10.1186/s13071-021-04877-1.
Article CAS PubMed PubMed Central Google Scholar
Sri-In C, Weng SC, Shiao SH, Tu WC. A simplified method for blood feeding, oral infection, and saliva collection of the dengue vector mosquitoes. PLoS ONE. 2020;15:e0233618. https://doi.org/10.1371/journal.pone.0233618.
Article CAS PubMed PubMed Central Google Scholar
Dong S, Balaraman V, Kantor AM, Lin J, Grant DG, Held NL, et al. Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion. PLoS Negl Trop Dis. 2017;11:e0005976. https://doi.org/10.1371/journal.pntd.0005976.
Article PubMed PubMed Central Google Scholar
Armstrong PM, Ehrlich HY, Magalhaes T, Miller MR, Conway PJ, Bransfield A, et al. Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential. Nat Microbiol. 2020;5:239–47. https://doi.org/10.1038/s41564-019-0619-y.
Article CAS PubMed Google Scholar
Vogels CBF, Ruckert C, Cavany SM, Perkins TA, Ebel GD, Grubaugh ND. Arbovirus coinfection and co-transmission: a neglected public health concern? PLoS Biol. 2019;17:e3000130. https://doi.org/10.1371/journal.pbio.3000130.
Article CAS PubMed PubMed Central Google Scholar
Goertz GP, Vogels CBF, Geertsema C, Koenraadt CJM, Pijlman GP. Mosquito co-infection with Zika and chikungunya virus allows simultaneous transmission without affecting vector competence of Aedes aegypti. PLoS Negl Trop Dis. 2017;11:e0005654. https://doi.org/10.1371/journal.pntd.0005654.
Article PubMed PubMed Central Google Scholar
Magalhaes T, Robison A, Young MC, Black WC, Foy BD, Ebel GD, et al. Sequential infection of Aedes aegypti mosquitoes with Chikungunya virus and Zika virus enhances early Zika virus transmission. Insects. 2018;9:4. https://doi.org/10.3390/insects9040177.
Article Google Scholar
Nuckols JT, Huang YJ, Higgs S, Miller AL, Pyles RB, Spratt HM, et al. Evaluation of simultaneous transmission of Chikungunya virus and Dengue virus type 2 in infected Aedes aegypti and Aedes albopictus (Diptera: Culicidae). J Med Entomol. 2015;52:447–51. https://doi.org/10.1093/jme/tjv017.
Article CAS PubMed PubMed Central Google Scholar
Dejnirattisai W, Jumnainsong A, Onsirisakul N, Fitton P, Vasanawathana S, Limpitikul W, et al. Cross-reacting antibodies enhance dengue virus infection in humans. Science. 2010;328:745–8. https://doi.org/10.1126/science.1185181.
Article CAS PubMed Google Scholar
Pantoja P, Perez-Guzman EX, Rodriguez IV, White LJ, Gonzalez O, Serrano C, et al. Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus. Nat Commun. 2017;8:15674. https://doi.org/10.1038/ncomms15674.
Article CAS PubMed PubMed Central Google Scholar
Bardina SV, Bunduc P, Tripathi S, Duehr J, Frere JJ, Brown JA, et al. Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity. Science. 2017;356:175–80. https://doi.org/10.1126/science.aal4365.
Article CAS PubMed PubMed Central Google Scholar
Senaratne UTN, Murugananthan K, Sirisena P, Carr JM, Noordeen F. Dengue virus co-infections with multiple serotypes do not result in a different clinical outcome compared to mono-infections. Epidemiol Infect. 2020;148:e119. https://doi.org/10.1017/S0950268820000229.
Article CAS PubMed PubMed Central Google Scholar
Li CJ, Huang PH, Chen HW, Chang SC. Development and characterization of mouse monoclonal antibodies targeting to distinct epitopes of Zika virus envelope protein for specific detection of Zika virus. Appl Microbiol Biotechnol. 2021;105:4663–73. https://doi.org/10.1007/s00253-021-11364-1.
Article CAS PubMed PubMed Central Google Scholar
Bosch I, de Puig H, Hiley M, Carre-Camps M, Perdomo-Celis F, Narvaez CF, et al. Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum. Sci Transl Med. 2017;9:409. https://doi.org/10.1126/scitranslmed.aan1589.
Article CAS Google Scholar
Rockstroh A, Moges B, Barzon L, Sinigaglia A, Palu G, Kumbukgolla W, et al. Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop. Emerg Microbes Infect. 2017;6:e99. https://doi.org/10.1038/emi.2017.87.
Article CAS PubMed PubMed Central Google Scholar
Amraoui F, Failloux AB. Chikungunya: an unexpected emergence in Europe. Curr Opin Virol. 2016;21:146–50. https://doi.org/10.1016/j.coviro.2016.09.014.
Article PubMed Google Scholar
Monteiro VVS, Navegantes-Lima KC, de Lemos AB, da Silva GL, de Souza GR, Reis JF, et al. Aedes-Chikungunya virus interaction: key role of vector midguts microbiota and its saliva in the host infection. Front Microbiol. 2019;10:492. https://doi.org/10.3389/fmicb.2019.00492.
Article PubMed PubMed Central Google Scholar
Caragata EP, Tikhe CV, Dimopoulos G. Curious entanglements: interactions between mosquitoes, their microbiota, and arboviruses. Curr Opin Virol. 2019;37:26–36. https://doi.org/10.1016/j.coviro.2019.05.005.
Article PubMed PubMed Central Google Scholar
Campbell CL, Harrison T, Hess AM, Ebel GD. MicroRNA levels are modulated in Aedes aegypti after exposure to Dengue-2. Insect Mol Biol. 2014;23:132–9. https://doi.org/10.1111/imb.12070.
Article CAS PubMed Google Scholar
Bryant B, Macdonald W, Raikhel AS. microRNA miR-275 is indispensable for blood digestion and egg development in the mosquito Aedes aegypti. Proc Natl Acad Sci USA. 2010;107:22391–8. https://doi.org/10.1073/pnas.1016230107.
Article PubMed PubMed Central Google Scholar
Eng MW, Clemons A, Hill C, Engel R, Severson DW, Behura SK. Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti. PLoS Negl Trop Dis. 2018;12:e0006186. https://doi.org/10.1371/journal.pntd.0006186.
Article CAS PubMed PubMed Central Google Scholar
Pompon J, Manuel M, Ng GK, Wong B, Shan C, Manokaran G, et al. Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to increase virus transmission. PLoS Pathog. 2017;13:e1006535. https://doi.org/10.1371/journal.ppat.1006535.
Article CAS PubMed PubMed Central Google Scholar
Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, et al. Dengue: a continuing global threat. Nat Rev Microbiol. 2010;8:S7-16. https://doi.org/10.1038/nrmicro2460.
Article CAS PubMed PubMed Central Google Scholar
Cologna R, Armstrong PM, Rico-Hesse R. Selection for virulent dengue viruses occurs in humans and mosquitoes. J Virol. 2005;79:853–9. https://doi.org/10.1128/JVI.79.2.853-859.2005.
Article CAS PubMed PubMed Central Google Scholar
King CC, Chao DY, Chien LJ, Chang GJ, Lin TH, Wu YC, et al. Comparative analysis of full genomic sequences among different genotypes of dengue virus type 3. Virol J. 2008;5:63. https://doi.org/10.1186/1743-422X-5-63.
Article CAS PubMed PubMed Central Google Scholar
Black WC IV, Bennett KE, Gorrochótegui-Escalante N, et al. Flavivirus susceptibility in Aedes aegypti. Arch Med Res. 2002;33:379–88. https://doi.org/10.1016/s0188-4409(02)00373-9.
Article CAS PubMed Google Scholar

Download references

Acknowledgements

We thank the Second Core Laboratory of the Department of Medical Research of National Taiwan University Hospital and the National Laboratory Animal Center for technical assistance. This work was supported by the Ministry of Science and Technology (Taiwan) Grant (MOST 109-2327-B-400-004) and (MOST 109-2320-B-002-062-MY3) to SHS, and NTUH UN109-018 and NTUH 112-UN0035 to PNT and SHS. This work was also supported by the National Taiwan University (NTU) Grant 111L2033-09 to SHS.

Funding

This work was supported by the Ministry of Science and Technology (Taiwan) Grant (MOST 109-2327-B-400-004) and (MOST 109-2320-B-002-062-MY3) to SHS, and NTUH UN109-018 and NTUH 112-UN0035 to PNT and SHS. This work was also supported by the National Taiwan University (NTU) Grant 111L2033-09 to SHS.

Author information

Daniel Chieh-Ding Lin and Shih-Che Weng contributed equally to this work.

Authors and Affiliations

Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, Taiwan
Daniel Chieh-Ding Lin
Department of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University, Taipei, Taiwan
Shih-Che Weng & Shin-Hong Shiao
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan
Po-Nien Tsao
Research Center for Developmental Biology & Regenerative Medicine, National Taiwan University, Taipei, Taiwan
Po-Nien Tsao
Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
Justin Jang Hann Chu

Authors

Daniel Chieh-Ding Lin
View author publications
You can also search for this author in PubMed Google Scholar
Shih-Che Weng
View author publications
You can also search for this author in PubMed Google Scholar
Po-Nien Tsao
View author publications
You can also search for this author in PubMed Google Scholar
Justin Jang Hann Chu
View author publications
You can also search for this author in PubMed Google Scholar
Shin-Hong Shiao
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

DCL, SCW, and SHS designed and performed the experiments, derived experimental models, and analyzed the data. PNT, JJHC, and SHS helped supervise the project. DCL, SCW, and SHS wrote the manuscript in consultation with PNT and JJHC. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shin-Hong Shiao.

Ethics declarations

Ethics approval and consent to participate

All animal procedures and experimental protocols were approved by an AAALAC-accredited facility and the Committee on the Ethics of Animal Experiments of the National Taiwan University College of Medicine (IACUC approval No. 20200210).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Figure S1. DENV2 and ZIKV single-infected cells are phenotypically distinct. A Cells inoculated with either DENV2 or ZIKV at MOI = 1 were stained for DAPIand flaviviral NS1at 2 dpi. B Cell density imaged under bright field revealed that DENV2-infected cells were less numerous than ZIKV-infected cells. Representative images shown.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Cite this article

Lin, D.CD., Weng, SC., Tsao, PN. et al. Co-infection of dengue and Zika viruses mutually enhances viral replication in the mosquito Aedes aegypti. Parasites Vectors 16, 160 (2023). https://doi.org/10.1186/s13071-023-05778-1

Download citation

Received: 21 January 2023
Accepted: 16 April 2023
Published: 11 May 2023
DOI: https://doi.org/10.1186/s13071-023-05778-1

Co-infection of dengue and Zika viruses mutually enhances viral replication in the mosquito Aedes aegypti