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Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus and a major cause of human viral encephalitis in Asia. We provide an overview of the knowledge on vector competence, vector capacity, and immunity of mosquitoes in relation to JEV. JEV has so far been detected in more than 30 mosquito species. This does not necessarily mean that these species contribute to JEV transmission under field conditions. Therefore, vector capacity, which considers vector competence, as well as environmental, behavioral, cellular, and biochemical variables, needs to be taken into account. Currently, 17 species can be considered as confirmed vectors for JEV and 10 other species as potential vectors. Culex tritaeniorhynchus and Culex annulirostris are considered primary JEV vectors in endemic regions. Culex pipiens and Aedes japonicus could be considered as potentially important vectors in the case of JEV introduction in new regions. Vector competence is determined by various factors, including vector immunity. The available knowledge on physical and physiological barriers, molecular pathways, antimicrobial peptides, and microbiome is discussed in detail. This review highlights that much remains to be studied about vector immunity against JEV in order to identify novel strategies to reduce JEV transmission by mosquitoes.

Background The incidence of human infections caused by arthropod-borne viruses, such as the chikungunya virus (CHIKV), has increased globally due to a number of factors, such as climate change and globalization. The exotic mosquito species Aedes albopictus is a significant vector for CHIKV, raising concerns about its transmission potential in temperate regions, including Central Europe. We have therefore investigated the vector competence of Ae. albopictus for CHIKV at constant and fluctuating temperatures between 15 °C and 24 °C to assess the transmission risk in Europe. Methods Aedes albopictus mosquitoes were reared and artificially infected with CHIKV. Infection rates and transmission efficiencies (TEs) were determined after 14 days of incubation at constant and fluctuating (± 5 °C) mean temperatures of 15 °C, 18 °C, 21 °C and 24 °C. In addition, mosquito locomotor activity was measured under the same fluctuating temperature conditions. A risk map for CHIKV transmission in Europe was generated combining temperature data and the current distribution of Ae. albopictus . Results CHIKV transmission was observed at all tested temperatures. The highest TEs were recorded at fluctuating temperatures of 18 °C (54.3%) and 21 °C (58.6%), while the lowest TE was observed at a constant temperature of 15 °C (5.6%). TEs at fluctuating temperatures of 15 °C and 24 °C were the same (32.5%). Mosquito activity showed a nocturnal unimodal activity pattern with a peak during the start of the scotophase (hour 20). The proportion of active mosquitoes per hour increased with temperature and was nearly zero at 15 °C. The risk map indicated that regions in Southern and Central Europe, including recently invaded areas north of the Alps, have temperatures theoretically allowing CHIKV transmission for at least some days per year. Conclusions While CHIKV can be transmitted by Ae. albopictus at 15 °C, the activity of this mosquito is strongly decreased at this temperature, likely reducing the transmission risk. These findings emphasize the importance of considering both vector competence and mosquito activity when assessing the risk of arbovirus transmission in temperate regions. Further studies are needed to validate these laboratory findings under field conditions. Graphical abstract

Background Japanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in Asia. Since the climate in some endemic countries is similar to temperate climates observed in Europe, understanding the role of specific mosquito species in the transmission of JEV is essential for predicting and effectively controlling the potential for the introduction and establishment of JEV in Europe. Methods This study aimed to investigate the vector competence of colonized Culex pipiens biotype molestus mosquitoes for JEV. The mosquitoes were initially collected from the field in southern Sweden. The mosquitoes were offered a blood meal containing the Nakayama strain of JEV (genotype III), and infection rates, dissemination rates, and transmission rates were evaluated at 14, 21, and 28 days post-feeding. Results The study revealed that colonized Swedish Cx. pipiens are susceptible to JEV infection, with a stable infection rate of around 10% at all timepoints. However, the virus was only detected in the legs of one mosquito at 21 days post-feeding, and no mosquito saliva contained JEV. Conclusions Overall, this research shows that Swedish Cx. pipiens can become infected with JEV, and emphasizes the importance of further understanding of the thresholds and barriers for JEV dissemination in mosquitoes. Graphical Abstract

Background The high prevalence of metabolic syndrome in low- and middle-income countries is linked to an increase in Western diet consumption, characterized by a high intake of processed foods, which impacts the levels of blood sugar and lipids, hormones, and cytokines. Hematophagous insect vectors, such as the yellow fever mosquito Aedes aegypti , rely on blood meals for reproduction and development and are therefore exposed to the components of blood plasma. However, the impact of the alteration of blood composition due to malnutrition and metabolic conditions on mosquito biology remains understudied. Methods In this study, we investigated the impact of whole-blood alterations resulting from a Western-type diet on the biology of Ae. aegypti . We kept C57Bl6/J mice on a high-fat, high-sucrose (HFHS) diet for 20 weeks and followed biological parameters, including plasma insulin and lipid levels, insulin tolerance, and weight gain, to validate the development of metabolic syndrome. We further allowed Ae. aegypti mosquitoes to feed on mice and tracked how altered host blood composition modulated parameters of vector capacity. Results Our findings identified that HFHS-fed mice resulted in reduced mosquito longevity and increased fecundity upon mosquito feeding, which correlated with alteration in the gene expression profile of nutrient sensing and physiological and metabolic markers as studied up to several days after blood ingestion. Conclusions Our study provides new insights into the overall effect of alterations of blood components on mosquito biology and its implications for the transmission of infectious diseases in conditions where the frequency of Western diet-induced metabolic syndromes is becoming more frequent. These findings highlight the importance of addressing metabolic health to further understand the spread of mosquito-borne illnesses in endemic areas. Graphical Abstract

Background The time required for PCR detection of DNA in human blood meals in vector mosquitoes may vary, depending on the molecular markers used, based on the size and copy number of the amplicons. Detailed knowledge of the blood-feeding behavior of mosquito populations in nature is an essential component for evaluating their vectorial capacity and for assessing the roles of individual vertebrates as potential hosts involved in the transmission of vector-borne diseases. Methods Laboratory experiments were conducted to compare the time course of PCR detection of DNA in human blood meals from individual blood-fed Anopheles stephensi mosquitoes, using loci with different characteristics, including two mitochondrial DNA (mtDNA) genes, cytB (228 bp) and 16S ribosomal RNA (rRNA) (157 bp) and nuclear Alu -repeat elements (226 bp) at different time points after the blood meal. Results Human DNA was detectable up to 84–120 h post-blood-feeding, depending on the length and copy number of the loci. Our results suggest that 16S rRNA and Alu -repeat markers can be successfully recovered from human DNA up to 5 days post-blood-meal. The 16S rDNA and Alu -repeat loci have a significantly ( P  = 0.008) slower decline rate than the cytB locus. Median detection periods (T50) for the amplicons were 117, 113 and 86.4 h for Alu -repeat, 16S rDNA and cytB, respectively, suggesting an inverse linear relationship between amplicon size/copy number and digestion time. Conclusion This comparative study shows that the Alu -repeat locus is the most efficient marker for time-course identification of human DNA from blood meals in female mosquitoes. It is also a promising tool for determining the anthropophilic index (AI) or human blood index (HBI), i.e. the proportion of blood meals from humans, which is often reported as a relative measure of anthropophagy of different mosquito vectors, and hence a measure of the vector competence of mosquito species collected in the field. Graphical Abstract

More than 3550 species of mosquitoes are known worldwide, and only a fraction is involved in the transmission of arboviruses. Mosquitoes in sylvatic and semi-sylvatic habitats may rapidly adapt to urban parks and metropolitan environments, increasing human contact. Many of these mosquitoes have been found naturally infected with arboviruses from the Alphaviridae, Flaviviridae, and Bunyaviridae families, with many being the cause of medically important diseases. However, there is a gap in knowledge about the vector status of newly invasive species and their potential threat to human and domestic animal populations. Due to their rapid distribution, adaptation to urban environments, and anthropophilic habits, some neglected mosquito species may deserve more attention regarding their role as secondary vectors. Taking these factors into account, we focus here on Aedes (Ochlerotatus) scapularis (Rondani), Aedes japonicus japonicus (Theobald), and Aedes (Fredwardsius) vittatus (Bigot) as species that have the potential to become important disease vectors. We further discuss the importance of these neglected mosquitoes and how factors such as urbanization, climate change, and globalization profoundly alter the dynamics of disease transmission and may increase the participation of neglected species in propagating diseases.

Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.

Aedes aegypti control programs have failed to restrain mosquito population expansion and, consequently, the spread of diseases such as dengue, Zika, and Chikungunya. Wolbachia infection of mosquitoes is a new and promising complementary tool for the control of arbovirus transmission. The use of Wolbachia-infected mosquitoes, mass reared using human blood, is currently being tested in several countries. However, the use of human blood for mass rearing mosquitoes, and thus expansion of this strategy, is problematic. With the aim of overcoming this problem, we tested the effect of different types of blood source on the fitness parameters of female Ae. aegypti and the Wolbachia titer over generations to be able to guarantee the suitability of an alternative source to human blood for mass rearing Wolbachia-infected mosquitoes. We investigated and compared essential parameters of the vector capacity of laboratory strains of Ae. aegypti with and without Wolbachia that fed on blood of different types of host (human, guinea pig, and mouse). The parameters analyzed were fecundity, fertility, pupation dynamics, and adult survival. Also, we tested whether it is possible to maintain mosquitoes with Wolbachia on mouse blood over generations without losing the bacterium titer. The average number of eggs per female, egg viability and pupation dynamics in the Wolbachia-infected mosquito (wMelBr) strain were similar, regardless of the blood source. The F1 progenies of females that fed on mouse blood or human blood were analyzed. The longevity of males was lower than that of females. F1 female survival differed depending on the presence of Wolbachia in the mother. In subsequent generations analyzed up until F35, the relative Wolbachia density was even higher when mosquitoes fed on mouse blood in comparison to human blood. Taken together, our results provide no evidence that the different types of blood influenced the fitness of the Wolbachia-infected mosquitoes. The presence of the bacterium in the colonies of Wolbachia-infected Ae. aegypti after 35 generations under the conditions evaluated indicates that they can be maintained on mouse blood. Based on these results, we show that it is possible to use mouse blood to feed female mosquitoes when using human blood for this purpose is problematic.

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted mainly by Lutzomyia longipalpis. However, canine cases have been reported in the absence of this species in the Greater São Paulo region, where Pintomyia fischeri and Migonemyia migonei are the predominant species. This raises the suspicion that they could be acting as vectors. Therefore, this study sought to investigate specific vector capacity parameters of these species and to compare them with those of Lu. longipalpis s.l. Among these parameters the blood feeding rate, the survival, and the susceptibility to the development of Le. infantum were evaluated for the three species, and the attractiveness of dogs to Pi. fischeri and Mg. migonei was evaluated. The estimated interval between blood meals was shorter for Lu. longipalpis s.l, followed by Pi. fischeri and Mg. migonei. The infection rate with Le. infantum flagellates in Lu. longipalpis was 9.8%, in Pi. fischeri 4.8%, and in Mg. migonei nil. The respective infective life expectancies (days) of Lu. longipalpis, Mg. migonei, and Pi. fischeri were 2.4, 1.94, and 1.68. Both Pi. fischeri and Mg. migonei were captured in the kennel with a predominance (95%) of Pi. fischeri. Considering the great attractiveness of dogs to Pi. fischeri, its susceptibility to infection by Le. infantum, infective life expectancies, and predominance in Greater São Paulo, this study presents evidence of Pi. fischeri as a potential vector of this parasite in the region.