Mosquitoes are more likely to acquire the dengue virus when they feed on blood with low levels of iron, researchers report in the 16 September issue of Nature Microbiology.
Supplementing people’s diets with iron in places where both iron deficiency anemia and dengue fever are a problem could potentially limit transmission of the disease, but there are risks.
Dengue fever is a disease spread by mosquitoes in the tropics, primarily Central America and northern South America, the Caribbean, sub-Saharan Africa and southeast Asia.
It has also been transmitted in the southeastern US. Dengue causes a fever, rash, and terrible aches, and can also lead to shock and death.
It causes about 60 million cases a year, with 18% requiring hospitalization and about 13,600 deaths, and costs about $9 billion annually worldwide.
Dengue is most commonly acquired in urban environments, and the expansion of cities in the tropics has been accompanied by an expansion in dengue infections.
A vaccine exists, but it can actually make the disease worse if given to someone who has never been previously infected.
Public health officials are actively looking for ways to reduce the prevalence of the disease.
UConn Health immunologist Penghua Wang wanted to see if blood quality had an impact on the spread of dengue virus.
Blood levels of various substances can vary tremendously from person to person, even among healthy people.
Wang and colleagues at Tsinghua University and State Key Laboratory of Infectious Disease Prevention and Control in Beijing, King Mongkut’s Institute of Technology Ladkrabang in Bangkok, and the 920 Hospital Joint Logistics Support Force in Kunming ran a series of experiments to explore the idea.
They collected fresh blood from healthy human volunteers, then added dengue virus to each sample.
Then they fed the blood to mosquitoes, and checked how many mosquitoes were infected from each batch.
They found it varied quite a lot. And the variation correlated very closely with the level of iron in the blood.
“The more iron in the blood, the fewer mosquitoes were infected,” says Wang. The team found it held true in a mouse model, too: mosquitoes feeding on mice infected with dengue were much more likely to acquire the virus if the mice were anemic.
The reason has to do with the mosquitoes’ own immune systems.
Cells in a mosquito’s gut take up iron in the blood and use it to produce reactive oxygen. The reactive oxygen kills the dengue virus.
“In areas where dengue is endemic, iron deficiency is more common.
It doesn’t necessarily explain it, the high prevalence of dengue…but it could be possible that iron supplementation could reduce dengue transmission to mosquitoes in those areas,” Wang says. But there’s a big caveat.
Malaria tends to be common in the same areas as dengue.
And plasmodium, the microorganism that causes malaria, thrives in an iron rich environment and might actually worsen if everyone is supplementing with iron.
Public health authorities need to weigh the costs and benefits before embarking on any population-wide supplementation program.
In any case, Wang says, understanding how dengue is transmitted will help public health authorities and scientists develop new ways to control the disease, and hopefully similar viruses such as Zika and West Nile virus as well.
Wolbachia’s ability to restrict arbovirus transmission makes it a promising tool to combat mosquito-transmitted diseases.
Wolbachia-infected Aedes aegypti are currently being released in locations such as Brazil, which regularly experience concurrent outbreaks of different arboviruses. A. aegypti can become co-infected with, and transmit multiple arboviruses with one bite, which can complicate patient diagnosis and treatment.
Methodology/principle findings
Using experimental oral infection of A. aegypti and then RT-qPCR, we examined ZIKV/DENV-1 and ZIKV/DENV-3 co-infection in Wolbachia-infected A. aegypti and observed that Wolbachia-infected mosquitoes experienced lower prevalence of infection and viral load than wildtype mosquitoes, even with an extra infecting virus. Critically, ZIKV/DENV co-infection had no significant impact on Wolbachia’s ability to reduce viral transmission. Wolbachia infection also strongly altered expression levels of key immune genes Defensin C and Transferrin 1, in a virus-dependent manner.
Conclusions/significance
Our results suggest that pathogen interference in Wolbachia-infected A. aegypti is not adversely affected by ZIKV/DENV co-infection, which suggests that Wolbachia-infected A. aegypti will likely prove suitable for controlling mosquito-borne diseases in environments with complex patterns of arbovirus transmission.
Author summary
Wolbachia is an endosymbiotic bacterium that infects insects. It has been artificially transferred into Aedes aegypti, a mosquito species that can transmit medically important viruses including dengue, chikungunya, and Zika. Wolbachia in A. aegypti limits infection with these viruses, making the mosquitoes much less capable of transmitting them to people.
In tropical areas, where these viral pathogens are commonly found, it is not unusual for outbreaks of different viruses to occur at the same time, which can complicate diagnosis and treatment for those afflicted.
Mosquitoes with Wolbachia are currently being released into these areas to reduce transmission of these diseases.
In our study, we assessed whether Wolbachia infection in A. aegypti mosquitoes could still effectively inhibit the dengue and Zika viruses if the mosquitoes were fed both viruses at the same time.
We found that Wolbachia was still very effective at inhibiting the replication of both viruses in the mosquito, and likewise still greatly reduced the chance of transmission of either virus. Our results suggest that Wolbachia-infected mosquitoes should be able to limit infection with more than one virus, should they encounter them in the field.
Introduction
Arboviruses, viruses transmitted by arthropods including Aedes mosquitoes, cause diseases that represent a serious threat to human health, with more than half of the global population at risk [1, 2]. Evidence suggests that the impact of these diseases is growing, as there has been an increase in case numbers of established arboviral infections [1, 3–5], while several other arboviruses have emerged as potential disease agents of the future [6, 7]. Consequently, finding a means to reduce the impact of these diseases is a significant public health issue.
The most significant viral pathogen transmitted by mosquitoes is dengue virus (DENV), which can cause severe fever, and produces an estimated 390 million infections per year [1]. DENV is highly prevalent in Latin America, and in South and Southeast Asia [3]. There are four known DENV serotypes (DENV-1 through DENV-4), and a subsequent infection with a second serotype can produce far more severe symptoms [1, 8]. This is particularly problematic, given that the multiple serotypes often co-circulate [9–11]. Compounding this issue is the lack of adequate treatment for dengue infections. There is no antiviral therapy, and while a vaccine effective against all four serotypes has been involved in late-stage clinical trials, the results have proven controversial [12, 13].
Another notable arbovirus is Zika virus (ZIKV), which was first isolated from febrile sentinel monkeys in 1947, in the Ziika forest of Uganda, and then emerged from relative obscurity to cause a massive outbreak that peaked during 2015–2016 [14]. The majority of cases of ZIKV appear to be asymptomatic, however infection has been associated with significant clinical consequences, including microcephaly and Guillain-Barré syndrome [15–17].
The mosquito Aedes aegypti is an important vector of arboviruses including DENV, chikungunya virus (CHIKV), yellow fever virus (YFV), and ZIKV. It has a broad distribution across tropical and sub-tropical regions around the world, where it lives in close association with human dwellings, and typically lays eggs in discarded containers that collect rainwater [18, 19]. Adult female A. aegypti feed on human blood in order to produce eggs, and they can become infected with an arbovirus after biting someone who is viremic. Within the mosquito, the virus invades the cells of the midgut epithelial layer where it exploits a range of host factors in order to replicate, and then releases mature virions that eventually infect the salivary glands [20–22]. This process takes approximately 7–14 days, depending on intrinsic and extrinsic factors including mosquito genetic background and environmental temperature, after which the mosquito can transmit the virus to new people when it bites them.
With the exception of yellow fever and Japanese Encephalitis virus, there are no effective commercially available vaccines against mosquito-borne arboviral diseases, and there are few viable treatment options available for those infected [1, 2]. For these reasons, disease prevention strategies are usually focused on mosquito control, relying on population elimination by clearing breeding sites, and using chemical insecticides to quickly and effectively kill mosquitoes [23]. However, maintaining persistent control of mosquito populations is a difficult and costly prospect, which has led to the development of many novel mosquito control strategies [24].
One such strategy involves Wolbachia pipientis, a maternally inherited bacterial endosymbiont of arthropods and worms that naturally occurs in at least 40% of terrestrial insect species [25]. Stable, heritable Wolbachia infections in A. aegypti have been generated by transinfection, the injection of Wolbachia from a donor species into A. aegypti eggs [26–29]. These Wolbachia-infected mosquitoes have been deployed in the field in multiple countries that experience high, endemic DENV transmission, as part of the World Mosquito Program (https://www.worldmosquitoprogram.org) [30–32].
Wolbachia is suitable for the biological control of mosquitoes because infection alters mosquito physiology and reproductive biology in ways that make them less effective vectors, facilitating the spread of the bacteria into wild populations [30, 33]. This ability to spread is due to cytoplasmic incompatibility, a reproductive manipulation that acts as a natural form of genetic drive specific to Wolbachia-infected insects [34, 35]. Cytoplasmic incompatibility occurs because the mating of Wolbachia-infected males to Wolbachia–uninfected females does not result in viable progeny. In contrast, Wolbachia-infected females produce viable, Wolbachia-infected progeny when mating to any male, thereby, proportionally increasing the number of Wolbachia-infected individuals.
Infection with some Wolbachia strains causes pathogen blocking—the restriction of viral infection and replication in the tissues and reduced likelihood of transmission in Wolbachia-infected mosquitoes. Wolbachia in A. aegypti strongly inhibits infection with viruses harmful to humans such as DENV, CHIKV, Mayaro virus, West Nile virus, YFV, and ZIKV [28, 36–42], and has been demonstrated to make them less effective vectors for a range of medically important mosquito-transmitted viruses [43, 44]. Multiple interactions between the mosquito immune system and Wolbachia have been characterized, however it is still unclear if any of these are definitively involved in virus blocking [33, 45]. Wolbachia may stimulate the host immune system to respond more effectively to viral infection, as the endosymbiont causes broad-spectrum immune induction [46–48]. It notably increases the expression of many immune genes, including antimicrobial peptides [47–50]. It also induces the production of reactive oxygen species, a key factor in antimicrobial immunity [47]. There are also putative links between Wolbachia and the mosquito RNAi immune pathway, through the gene Argonaute 2 [51]. Wolbachia infection alters miRNA production, and the subsequent changes to gene expression could impact on viral infection [52]. It causes the downregulation of an insulin receptor that is linked to DENV infection [53]. Finally, there is evidence that Wolbachia preferentially utilizes host resources, like cholesterol, that are also required for viral infection [54, 55].
Releases of A. aegypti mosquitoes infected by the wMel Wolbachia strain have been ongoing in Rio de Janeiro, Brazil since 2014 (https://tinyurl.com/WMPBrazil, [56]. Mosquito control strategies in disease-endemic regions, including Rio de Janeiro, may be complicated by the co-circulation of different arboviruses [9, 57]. Multiple studies have demonstrated that humans can become infected with more than one mosquito-transmitted virus at a time, complicating diagnosis and recovery [10, 58]. Recent studies have also determined that A. aegypti can harbour and transmit more than one virus, after experimental co-infection, suggesting that a single mosquito bite could lead to a person contracting two diseases [59, 60]. Interestingly, co-infection may lead to competition between viruses, producing differential infection rates, and even altering transmission rates. For instance, during co-infection with ZIKV/DENV-2, the transmission of ZIKV is favoured [59]. Other studies have shown that co-infection can promote viral infection and transmission. For example, sequential infection with ZIKV and CHIKV enhances ZIKV transmission in A. aegypti [61], and co-infection of CHIKV/DENV-2 leads to increased replication of DENV, compared to mono-infection [62]. Little is known about the impact of co-infection on the mosquito immune system, however there are differences in the host proteomic profile for CHIKV/DENV co-infected and mono-infected mosquitoes [63], which suggests that there is some impact.
However, to the best of our knowledge, no studies have described the effects of viral co-infection in Wolbachia-infected mosquitoes. Given that the mechanism of Wolbachia-induced pathogen blocking may be immune based, we were interested to see how the bacterium responded to infection with multiple arboviruses. Any decrease of blocking activity would be particularly important given that Wolbachia-infected mosquitoes are currently being released into areas where multiple medically important arboviruses are transmitted. For these reasons, we sought to determine whether co-infection with ZIKV and DENV-1, or with ZIKV and DENV-3, all recently circulating in Brazil, would lead to less effective pathogen blocking in the Wolbachia-infected A. aegypti line that is currently being used for mosquito control in that country, as well as in other parts of the world. To do so, we orally challenged Wolbachia-infected and -uninfected mosquitoes with ZIKV/DENV-1 or ZIKV/DENV-3, and then examined disseminated infection rates and viral load in mosquito heads and thoraces at 7, 14, and 21 days post-infection (dpi). We also examined the effect on virus transmission, using saliva samples collected at 14 and 21dpi.
To gain further insight into the nature of interactions between Wolbachia, arboviruses and the mosquito immune system, we examined the effect of Wolbachia infection, and ZIKV/DENV co-infection on five candidate genes that had previously been linked to Wolbachia-induced viral interference and/or mosquito response to viral infection. We selected two genes that had previously been associated with Wolbachia infection and virus interference: Defensin C (DEFC), an antimicrobial peptide that is upregulated during Wolbachia infection [47–49], and Transferrin 1 (TSF), an iron-binding protein with a putative immune role, which is upregulated during Wolbachia infection [48, 49]. We selected Niemann-Pick Protein C1b (NPC1b), a cholesterol binding protein involved in DENV infection in A. aegypti [64], due to the known association between Wolbachia, viruses and host cholesterol [54, 55]. Finally, we selected two genes that were previously identified as being responsive to viral infection in A. aegypti but were not characterized in Wolbachia-infected mosquitoes: a pupal cuticle protein (PCP) that is downregulated during DENV infection in A. aegypti [65], and a putative NF-κB repressing factor (NFKBR) that is upregulated during arboviral infection but downregulated by Wolbachia [48, 65]. We observed that all of these genes were differentially expressed due to Wolbachia infection, ZIKV and/or DENV infection, or Wolbachia x virus infection status.
Our results indicated that very few Wolbachia-infected mosquitoes become infected by either ZIKV or DENV after experimental co-infection with either ZIKV/DENV-1 or ZIKV/DENV-3, which suggests that Wolbachia-induced pathogen interference is effective against multiple pathogens. Our observation that Wolbachia-induced inhibition of co-infecting viruses extended to the level of transmission suggests that Wolbachia-infected mosquitoes are likely to be a suitable tool for disease control in areas experiencing simultaneous transmission of multiple arboviruses.
More information: Yibin Zhu et al. Host serum iron modulates dengue virus acquisition by mosquitoes. Nature Microbiology (2019) DOI: 10.1038/s41564-019-0555-x
Journal information: Nature Microbiology
Provided by University of Connecticut
