Uncertainties Surrounding Madariaga Virus, a Member of the Eastern Equine Encephalitis Virus Complex.

Background: Madariaga virus (MADV), a member of the eastern equine encephalitis virus (EEEV) complex, circulates in Latin America and exhibits distinct evolutionary and ecological features compared to the North American EEEV. While published data have shed light on MADV ecology, several key aspects remain unknown. Methods: In this study, we compiled data on virus isolation, vector competence, and animal serology collected over six decades in Latin America to identify critical knowledge gaps on MADV transmission and ecology. Results: Specific vertebrate animals serving as amplifying hosts and the mosquito species acting as enzootic and epizootic vectors have not yet been identified. Other aspects that remain unclear are the virus current geographic distribution, the role of equines as hosts in epizootic cycles, and the full impact of MADV on human health in endemic regions. Conclusions: The numerous knowledge gaps surrounding MADV, its widespread distribution in Latin America, and its potential to cause severe disease in animals and humans emphasize the urgent need for increased research efforts, heightened awareness, and intensified surveillance towards this potential emerging threat.

A comparative analysis of Aedes albopictus and Aedes aegypti subjected to diapause-inducing conditions reveals conserved and divergent aspects associated with diapause, as well as novel genes associated with its onset.

Aedes albopictus and Aedes aegypti are mosquito species that are distributed worldwide and transmit diverse arboviruses of medical importance, such as those causing yellow fever, dengue, chikungunya and Zika. A. albopictus embryos may remain viable for long periods in the environment due to their ability to become dormant through quiescence or diapause, a feature that contributes to their dispersion and hinders control actions. Diapause incidence can vary among natural populations of A. albopictus, but metabolic and genetic parameters associated with its induction still need to be better defined. The present study aimed to investigate the effect of exposure to diapause-inducing conditions on several biological parameters in different populations of A. albopictus (from tropical and temperate areas) and the diapause-refractory A. aegypti (tropical and subtropical populations). As expected, only the A. albopictus populations exhibited diapause, but with a lower incidence for the population from a tropical area. Exposure to diapause-inducing conditions, however, led to a sharp reduction in fecundity for both A. albopictus and A. aegypti tropical populations, with no effect on fertility (>90%). It also led to a prolonged period as pupae for the progeny of all induced groups, with a further delay for those from temperate climates. In all those induced groups, the lipid contents in eggs and adult females were higher than in the non-induced controls, with the highest values observed for both A. albopictus groups. Three genes were selected to have their expression profile investigated: cathepsin, idgf4, and pepck. Upon exposure to diapause-inducing conditions, all three genes were upregulated in the A. albopictus embryos from the tropical region, but only idgf4 was upregulated in the temperate climate embryos. This represents a new gene associated with diapause that can be used as a target to evaluate and prevent embryonic dormancy, a possible new vector control strategy for mosquito species from temperate areas, such as A. albopictus.

Polymorphisms in GSTE2 is associated with temephos resistance in Aedes aegypti.

The glutathione S-transferases (GSTs) are enzymes involved in several distinct biological processes. In insects, the GSTs, especially delta and epsilon classes, play a key role in the metabolism of xenobiotics used to control insect populations. Here, we investigated its potential role in temephos resistance, examining the GSTE2 gene from susceptible (RecL) and resistant (RecR) strains of the mosquito Aedes aegypti, vector for several pathogenic arboviruses. Total GST enzymatic activity and the GSTE2 gene expression profile were evaluated, with the GSTE2 cDNA and genomic loci sequenced from both strains. Recombinant GSTE2 and mutants were produced in a heterologous expression system and assayed for enzyme kinetic parameters. These proteins also had their 3D structure predicted through molecular modeling. Our results showed that RecR has a profile of total GST enzymatic activity higher than RecL, with the expression of the GSTE2 gene in resistant larvae increasing six folds. Four exclusive RecR mutations were observed (L111S, I150V, E178A and A198E), which were absent in the laboratory susceptible strains. The enzymatic activity of the recombinant GSTE2 showed different kinetic parameters, with the GSTE2 RecR showing an enhanced ability to metabolize its substrate. The I150V mutation was shown to induce significant changes in catalytic parameters and a 3D modeling of GSTE2 mapped two of the RecR changes (L111S and I150V) near the enzyme's catalytic pocket, also implying an impact on its catalytic activity. Our results reinforce a potential role for GSTE2 in the metabolic resistance phenotype while contributing to the understanding of the molecular basis for the resistance mechanism.

Fitness cost in field and laboratory Aedes aegypti populations associated with resistance to the insecticide temephos.

BACKGROUND: The continued use of chemical insecticides in the context of the National Program of Dengue Control in Brazil has generated a high selective pressure on the natural populations of Aedes aegypti, leading to their resistance to these compounds in the field. Fitness costs have been described as adaptive consequences of resistance. This study evaluated the biological and reproductive performance of A. aegypti strains and a field population resistant to temephos, the main larvicide used for controlling mosquitoes. METHODS: Comparative tests were performed with a resistant field population from the municipality of Arcoverde, Pernambuco State, Brazil, with a high rate of temephos resistance (RR = 226.6) and three isogenetic laboratory strains from the same origin (Araripina municipality, Pernambuco): RecR (RR = 283.6); RecRNEx (RR = 250.5), a strain under a process of resistance reversion; and RecRev (RR = 2.32), a reversed susceptible strain used as an experimental control. RESULTS: Our study revealed that the absence of selective pressure imposed by exposure to temephos, for five consecutive generations, led to a discrete reduction of the resistance ratio and the response of the detoxifying enzymes. Most of the 19 biological parameters were impaired in the resistant strains and field population. The analysis of the fertility life table confirmed the presence of reproductive disadvantages for the resistant individuals. Similarly, the longevity, body size, and total energetic resources were also lower for the resistant females, except for the last two parameters in the field females (Arcoverde). In contrast, the sex ratio and embryonic viability suffered no interference in all strains or population evaluated, regardless of their status of resistance to temephos. CONCLUSIONS: The reproductive potential and survival of the resistant individuals were compromised. The parameters most affected were the larval development time, fecundity, net reproduction rate, and the generational doubling time. These fitness costs in the natural population and laboratory strains investigated are likely associated with maintaining the metabolic mechanism of resistance to temephos. Our results show that despite these costs, the highly temephos resistant populations can compensate for these losses and successfully overcome the control actions that are based on the use of chemical insecticides.

Vector competence of the Aedes aegypti population from Santiago Island, Cape Verde, to different serotypes of dengue virus.

BACKGROUND: Dengue is an arboviral disease caused by dengue virus (DENV), whose main vectors are the mosquitoes Aedes aegypti and Aedes albopictus. A. aegypti is the only DENV vector in Cape Verde, an African country that suffered its first outbreak of dengue in 2009. However, little is known about the variation in the level of vector competence of this mosquito population to the different DENV serotypes. This study aimed to evaluate the vector competence of A. aegypti from the island of Santiago, Cape Verde, to four DENV serotypes and to detect DENV vertical transmission. METHODS: Mosquitoes were fed on blood containing DENV serotypes and were dissected at 7, 14 and 21 days post-infection (dpi) to detect the virus in the midgut, head and salivary glands (SG) using RT-PCR. Additionally, the number of copies of viral RNA present in the SG was determined by qRT-PCR. Furthermore, eggs were collected in the field and adult mosquitoes obtained were analyzed by RT-PCR and the platelia dengue NS1 antigen kit to detect transovarial transmission. RESULTS: High rates of SG infection were observed for DENV-2 and DENV-3 whereas for DENV-1, viral RNA was only detected in the midgut and head. DENV-4 did not spread to the head or SG, maintaining the infection only in the midgut. The number of viral RNA copies in the SG did not vary significantly between DENV-2 and DENV-3 or among the different periods of incubation and the various titers of DENV tested. With respect to DENV surveillance in mosquitoes obtained from the eggs collected in the field, no samples were positive. CONCLUSION: Although no DENV positive samples were collected from the field in 2014, it is important to highlight that the A. aegypti population from Santiago Islands exhibited different degrees of susceptibility to DENV serotypes. This population showed a high vector competence for DENV-2 and DENV-3 strains and a low susceptibility to DENV-1 and DENV-4. Viral RNA copies in the SG remained constant for at least 21 dpi, which may enhance the vector capacity of A. aegypti and suggests the presence of a mechanism modulating virus replication in the SG.