Vectorial Capacity Research Articles

Cotton leaf curl Multan virus activates autophagy in the whitefly AsiaII7, weakening its vectorial capacity for transmission.

Autophagy plays an important role against pathogen infections in both insects and plants. Insect vectors employ autophagy as an intrinsic antiviral defense mechanism against viral infections, whereas viruses can exploit autophagy to enhance their transmission via insect vectors. The Cotton leaf curl Multan virus (CLCuMuV) is transmitted by the AsiaII7 cryptic species of Bemisia tabaci, however, the role of autophagy is involved in regulating the transmission of this virus remains unclear. In this study, it was observed that CLCuMuV infection induced autophagy in AsiaII7 whitefly, as evidenced by an elevated in the level of ATG8-II and the upregulation of Atg3, Atg8, Atg9 and Atg12. Both the administration of the autophagy inhibitor bafilomycin A1 and the silencing of Atg9 expression increased the viral load and enhanced CLCuMuV transmission. Conversely, the activation of autophagy via rapamycin feeding significantly reduced the amount of CLCuMuV and inhibited the efficiency of virus transmission. CLCuMuV infection can activate the autophagy pathway in whiteflies. The activation of autophagy leads to the subsequent degradation of the virus and suppresses CLCuMuV transmission efficiency, whereas suppression of autophagy promotes virus transmission. Our research provides insight into the potential role of autophagy in antiviral defense mechanisms. © 2025 Society of Chemical Industry.

Reduction in Aedes aegypti Population After a Year-Long Application of Targeted Sterile Insect Releases in the West Valley Region of Southern California.

Aedes aegypti is of great public health concern because of its vectorial capacity to transmit various arboviruses such as Zika, yellow fever, dengue, and chikungunya. In California, its expanding geographic distribution has been unrestrained. This urgently calls for innovative tools such as the use of sterile insect technique (SIT) to strengthen invasive Aedes control. This study aimed to determine the efficacy of combining an SIT application into integrated vector management (IVM) strategies in invasive Aedes control in the West Valley region of southern California. A total of 25 Aedes hotspots, grouped into two cohorts-sites that received SIT treatment only (n = 9) and sites that received both SIT and In2Care® Mosquito Stations (n = 16)-were selected for this study. Biweekly, X-ray irradiated male Ae. aegypti mosquitoes were released between April and November 2024. Data from weekly BG Sentinel-2 traps were utilized to compare the mosquito densities between the pre-intervention (2023) and intervention (2024) periods for both cohorts. In addition, a subset of samples (n = 50) of irradiated male mosquitoes were placed in a separate cage with freshly emerged unirradiated female mosquitoes to evaluate the level of sterility of the eggs from these mosquitoes. Aedes-related service requests made by the District's residents were also compared between the pre-intervention and intervention periods to indirectly determine the effect of optimizing the control strategies on overall mosquito problems. A total of 106,608 sterile male Ae. aegypti were released between April and November 2024. The cohort with the SIT only application showed a 44% reduction in the number of female Ae. aegypti per trap-night during the intervention period compared with the pre-intervention period. At sites with In2Care Mosquito Stations that also received SIT treatment, the mosquito density dropped by 65% during the intervention period compared with the pre-intervention period. The number of Aedes-related service requests during the intervention year (n = 367) was 45% lower than the pre-intervention year (n = 656). Over 99.6% eggs collected from female mosquitoes mated with irradiated males did not hatch, indicating a high level of sterility of the irradiated males utilized for our SIT application. Here, we demonstrated the potential of SIT when combined with IVM strategies such as In2Care® Mosquito Stations, offering a holistic approach to reducing the public health risks associated with Aedes-borne diseases.

Occurrence Of Limatus Durhamii In Artificial Containers In Atlantic Forest, São Paulo, Brazil.

has wild habits with ecological plasticity in anthropized environments. We report the occurrence of this species in several types of artificial containers in 2 fragments of the Atlantic Forest in the city of São Paulo, Brazil. A total of 1,428 larvae were collected, in which plastic containers had the highest abundance of immature forms of Li. durhamii, with 579 (40.5%) specimens collected, followed by glass bottles with 464 (32.5%) and metal cans with 223 (15.6%). The high abundance of Li. durhamii in artificial containers points to an adaptation process to impacted environments. Therefore, studies that investigate the feeding habits and vectorial capacity of this species are essential to understanding its epidemiological role in the environment.

Assessing neighbourhood-scale BTI spray applications and laboratory-based mortality testing on Aedes aegypti larval development.

Mosquitoes, particularly Aedes aegypti, pose significant public health risks by transmitting diseases like dengue, zika and chikungunya. Bacillus thuringiensis var. israelensis (BTI) is a crucial larvicide targeting mosquitoes while sparing other organisms and the environment. This study evaluated the effects of sublethal BTI doses on Ae. aegypti larvae regarding mortality, development, adult emergence and size, using a wide-area spray application in an urban neighbourhood. Laboratory experiments with four BTI concentrations (0, 0.008, 0.02 and 0.04 ppm) assessed compensatory and over compensatory responses. The spray achieved over 90% larval mortality within 48 h, but accumulating sublethal doses could trigger compensatory and over compensatory effects, enhancing the fitness of survivors. A dose-response relationship was evident, with higher BTI concentrations leading to increased mortality, reduced longevity and fewer pupae. BTI exposure also skewed the sex ratio towards males and altered adult sizes, potentially affecting population dynamics and vectorial capacity. These findings highlight the effectiveness of BTI in Ae. aegypti control and the importance of understanding compensation, overcompensation and density-dependent effects. While wide-area BTI applications can reach inaccessible breeding sites and offer potent mosquito control, careful consideration of ecological and evolutionary consequences is crucial.

Integrated surveillance for Oropouche Virus: molecular evidence of potential urban vectors during an outbreak in the Brazilian Amazon

The Oropouche Virus (OROV) has Culicoides paraensis as its main vector in the urban cycle. Diagnoses of acute febrile cases and entomological collections were performed between January and April 2024 in the urban setting of Porto Velho, a city located in the western Brazilian Amazon. A total of 904 human samples were evaluated using RT-qPCR, of which 328 were positive for OROV. In addition, 3,950 insects were captured and Culex quinquefasciatus was the most abundant and predominant species. Seven mosquito samples showed amplification for OROV from pools of Cx. quinquefasciatus, Limatus durhamii and Aedes albopictus. Due to the first record of OROV in the two latter species, our findings can support new hypotheses about potential or bridge vectors. In turn, the high density of Cx. quinquefasciatus reinforces the supposition of this species as the main urban vector in the Amazon in the absence of C. paraensis. Complementary studies of vector competence and capacity are necessary for confirmation.

Indoor Residual Spraying With Fludora Fusion for Malaria Control in Pyrethroid‐Resistant Areas of Gujarat, India: A Community‐Randomized Trial

Background: The development of insecticide resistance in malaria vectors has necessitated a need to evaluate new insecticide molecules with different modes of action. In the present study, Fludora Fusion 562.5 WP‐SB (clothianidin 50% + deltamethrin 6.25% AI/kg) was evaluated for its efficacy and residual action for the control of pyrethroid‐resistant malaria vector, Anopheles culicifacies (Diptera: Culicidae), during May 2017 to February 2018 in Gujarat state, India.Methods: Fludora Fusion at the dose of 225 mg AI/m2 and bendiocarb at a dose of 400 mg AI/m2 as a positive control were sprayed in 5 villages each in districts of Kheda, Vadodara, and Panchmahal. The persistence of their efficacy on different local surfaces was determined against An. culicifacies. Entomological indices such as indoor resting density, human landing collections, pyrethrum spray collections, and exit trap collections were monitored to assess the impact of spraying.Results: The observed residual action of Fludora Fusion on mud and cement surfaces was for 6 months and bendiocarb for 3–4 months on both surfaces. Indoor resting densities and parous rate of An. culicifacies were significantly lower in houses sprayed with Fludora Fusion compared to bendiocarb‐sprayed houses. Daily entomological inoculation rate (EIR) declined from 1.275 during prespray period to 0.5225 in the Fludora Fusion arm and 0.3802 in the Ficam arm in postspray period, indicating a reduction in the malaria transmission potential of An. culicifacies in both arms.Conclusion: Based on the residual action of the Fludora Fusion on most common sprayed surfaces and its effects on the elements of vectorial capacity, Fludora Fusion at 225 mg/m2 dose was found effective for more than 6 months and could be a potential option for the control of resistant mosquito vectors.

The Interspecific Competition Between Larvae of Aedes aegypti and Major African Malaria Vectors in a Semi-Field System in Tanzania.

Interspecific competition between mosquito larvae may affects adult vectorial capacity, potentially reducing disease transmission. It also influences population dynamics, and cannibalistic and predatory behaviors. However, knowledge of interspecific competition between Ae. aegypti and Anopheles species is limited. The study examined interspecific competition between Ae. aegypti larvae and either An.arabiensis, An. gambiae, or An. funestus on individual fitness in semi-field settings. The experiments involved density combinations of 100:100, 200:0, and 0:200 (Ae. aegypti: Anopheles), reared with and without food, in small habitats (8.5 cm height × 15 cm diameter) with 0.5 liters of water and large habitats (15 cm height × 35 cm diameter) with 1 liter of water. The first group received Tetramin® fish food (0.02 g), while the second group was unfed to assess cannibalism and predation. While interspecific competition affected both genera, Anopheles species experienced greater effects, with reduced survival and delayed development, compared to Ae. aegypti. The mean wing lengths of all species were significantly small in small habitats with a mixed population (p < 0.001). The presence of food reduced cannibalism and predation compared to its absence. These interactions have implications for diseases transmission dynamics and can serve as biological indicators to signal the impacts of vector control interventions.

Automated age grading of female Culex pipiens by an optical sensor system coupled to a mosquito trap

BackgroundThe age distribution of a mosquito population is a major determinant of its vectorial capacity. To contribute to disease transmission, a competent mosquito vector, carrying a pathogen, must live longer than the extrinsic incubation period of that pathogen to enable transmission to a new host. As such, determining the age of female mosquitoes is of significant interest for vector-borne diseases surveillance and control programs.MethodsIn this contribution, an automated age-grading system was developed to classify the age of female Culex pipiens, which is the primary vector of West Nile virus and other pathogens of medical and veterinary importance in northern latitudes. The system comprises an optical wingbeat sensor coupled to the entrance of a mosquito trap and a machine learning model. Three age classes were used in the study: young (2–4 days), middle (7–9 days) and old (14–16 days). From a balanced dataset of flight data, four features were extracted: wingbeat fundamental frequency, spectrogram, power spectral density and Mel frequency cepstral coefficients. The features were used for training with the XGBoost algorithm to generate a model for age classification.ResultsThe best performing model was trained with the power spectral density feature on two age classes, ≤ 4 days old and ≥ 7 days old, and had an accuracy of 71.8%.ConclusionsAn automated mosquito age-grading system was applied for the first time to our knowledge for automated age classification in mosquitoes; and complements the mosquito genus and sex classification capability of the system reported in our previous work. The system may find use in mosquito-borne disease surveillance and control to help to discriminate young mosquitoes (≤ 4 days old) from older mosquitoes, which may act as vectors of arboviruses.Graphical

Genomic analyses revealed low genetic variation in the intron-exon boundary of the doublesex gene within the natural populations of An. gambiae s.l. in Burkina Faso

BackgroundThe recent success of a population control gene drive targeting the doublesex gene in Anopheles gambiae paved the way for developing self-sustaining and self-limiting genetic control strategies targeting the sex determination pathway to reduce and/or distort the reproductive capacity of insect vectors. However, targeting these genes for genetic control requires a better understanding of their genetic variation in natural populations to ensure effective gene drive spread. Using whole genome sequencing (WGS) data from the Ag1000G project (Ag3.0, 3.4 and 3.8), and Illumina pooled amplicon sequencing, we investigated the genetic polymorphism of the intron-4–exon-5 boundary of the doublesex gene in the natural populations of An. gambiae sensu lato (s.l.).ResultsThe analyses showed a very low variant density at the gRNA target sequence of the Ag(QFS)1 gene drive (previously called dsxFCRISPRh) within the populations of West and East Africa. However, populations from the forest area in Central Africa exhibited four SNP at frequencies ranging from 0.011 to 0.26. The SNP (2R:48714641[C > T]) at high frequencies, i.e. 0.26 is identified within the An. coluzzii population from Angola. The analyses also identified 90 low frequency (1 − 5%) SNPs in the genomic region around the gRNA target sequence (intron-4–exon-5 boundary). Three of these SNPs (2R:48714472 A > T; 2R:48714486 C > A; 2R:48714516 C > T) were observed at frequencies higher than 5% in the UTR region of the doublesex gene. The results also showed a very low variant density and constant nucleotide diversity over a five-year survey in natural An. gambiae s.l. populations of Burkina Faso.ConclusionThese findings will guide the implementation of doublesex-targeted gene drives to support the current control tools in malaria elimination efforts. Our methods can be applied to efficiently monitor the evolution of any sequence of interest in a natural population via pooled amplicon sequencing, surpassing the need for WGS.

The effect of novel mosquito bite prevention tools on Anopheles minimus landing and key secondary endpoints: semi-field evaluations in Thailand

BackgroundThe Greater Mekong Subregion (GMS) aims to eliminate all human malaria by 2030 and is making substantial progress toward this goal, with malaria increasingly confined to forest foci. These transmission foci are predominantly inhabited by ethnic minorities, local populations, and rural mobile and migrant populations working in mining and agriculture. The recommendations of the World Health Organization (WHO) on malaria elimination states that small population groups which constitute a large proportion of the malaria transmission reservoir should benefit from targeted strategies to reduce transmission overall. These population groups are exposed to malaria vector bites during the day due to Anopheles daytime biting, and during the night, due to low bed net use and open sleeping structures. Such characteristics limit the effectiveness of the WHO core vector control strategies [indoor residual spraying (IRS), insecticide-treated nets (ITNs)], which target indoor resting and indoor feeding mosquitoes. Interventions that target daytime and outdoor resting or biting mosquitoes, and which complement IRS and ITNs and drug strategies, may hasten a decline in the malaria burden.MethodsThis study evaluated two transfluthrin- and one metofluthrin-based volatile pyrethroid spatial repellents (VPSRs), and etofenprox insecticide-treated clothing (ITC) with and without a topical repellent in a semi-field system (SFS) at two research sites in Thailand, across two trial rounds. The study estimated the protective efficacies of the vector control tools against two pyrethroid-susceptible Anopheles minimus strains in the form of 15 interventions, including a combined VPSR and ITC intervention. The interventions’ modes of action were studied by measuring their impact on mosquito landing, and on key life history traits known to affect vectoral capacity (knockdown, post-exposure blood feeding, and 24-h mortality) using a block-randomized crossover design. The odds ratio (OR) for each intervention compared to the control on each outcome was estimated.ResultsAll interventions substantially reduced An. minimus landings and prevented more than 50% mosquito landings when new (VPSRs) or unwashed (treated clothing). In addition to landing reduction, all interventions decreased post-exposure blood feeding, induced knockdown and increased mortality at 24 h. The VPSR interventions were generally more protective against landing than the treated clothing intervention. The combined intervention (VPSR + ITC) provided the greatest protection overall.ConclusionThis SFS evaluation indicates an effect of these VPSR and ITC interventions in reducing An. minimus landing for the user, and indicates their potential for community protection by secondary modes of action. This study demonstrates the utility of SFS trials in the evaluation of bite prevention tools and emphasizes the need for multiple evaluations at different sites. It also highlights possible sources of biases observed, including the measuring of mosquito landing rather than biting, weather parameters, and low mosquito recapture.

Heat stress promotes the accumulation of tomato yellow leaf curl virus in its insect vector by activating heat shock factor

High temperature tends to be a contributing factor to the spread of vector-borne viral diseases by enhancing vector competence. However, the underlying mechanisms of the increased virus transmission capacity of vectors under heat stress are still largely unknown. Tomato yellow leaf curl virus (TYLCV) is one of the most devastating plant DNA viruses worldwide and is transmitted exclusively by Bemisia tabaci. Here, we identified heat shock factor (HSF) as a key transcription factor that mediates TYLCV accumulation in whitefly vectors under heat stress. Quantitative analysis revealed that the amount of TYLCV DNA in whiteflies gradually increased with increasing temperature (above 38 °C). To determine the underlying mechanism, RNA-seq analysis was performed, which revealed that 1 h of heat stress caused a dramatic increase in heat shock protein (HSP) expression. Moreover, the whitefly HSP transcription factor HSF was shown to specifically interact with the intergenic region of TYLCV via yeast one-hybrid and dual-luciferase analyses. Additionally, the transcriptional activity of HSF gradually increased with increasing heat treatment temperature, indicating a similar pattern of virus accumulation under heat stress. Knocking down HSF in whiteflies caused a significant decrease in TYLCV DNA and coat protein. Our results reveal the important role of HSF in mediating virus accumulation in insect vectors under high temperature and provide insights into how environmental factors affect plant virus‒vector interactions.

Blood meal source of field-caught &lt;em&gt;Anopheles &lt;/em&gt;vector mosquitoes of malaria from Northern Sri Lanka

Host preference in blood-feeding anopheline mosquitoes is crucial in determining malaria vectors. Using a PCR assay that targeted a portion of the Cytochrome b gene of five vertebrates, this study sought to investigate the blood meals of 30 field-caught Anopheles mosquitoes from two Northern Districts of Sri Lanka. Study revealed that blood meals of Anopheles culicifacies and Anopheles subpictus mosquitoes were exclusively from humans (23.3%) and goats (50%), and the remaining 26.6% were mixed blood meals. An. culicifacies (0.65) was more anthropophilic than An. subpictus (0.31), based on the Human Blood Index. Single-host blood meals were more common in both An. culicifacies (70.6%) and An. subpictus (76.92%) than multiple-host blood meals. A significant association (p&lt;0.05) was observed between the vertebrate source of the blood meal of the mosquito and the frequency of single and multiple-host blood meals taken. While the conventional multiplex PCR assay was more successful in identifying the source of multiple-host blood meals, the nested PCR technique effectively amplified the DNA of single-host blood meals. The mean DNA concentration of mixed-host blood meal extractions amplified by conventional PCR assay (121.8±37.3 ng/µL) was substantially greater (p&lt;0.05) than that of the nested PCR (39.5±29.7 ng/µL). This study reveals important findings which are significant for further exploration into the vectorial capacity of An. culicifacies and An. subpictus, and consequently their transmission potential as malaria vectors in the area. This understanding of the feeding patterns of Anopheles vectors will be helpful for long-term planning and development of suitable, situation-specific malaria control measures in a country.

Investigating the Effects of Microclimate on Arboviral Kinetics in Aedes aegypti

Aedes aegypti are indoor-dwelling vectors of many arboviruses, including Zika (ZIKV) and chikungunya (CHIKV). The dynamics of these viruses within the mosquito are known to be temperature-dependent, and models that address risk and predictions of the transmission efficiency and patterns typically use meteorological temperature data. These data do not differentiate the temperatures experienced by mosquitoes in different microclimates, such as indoor vs. outdoor. Using temperature data collected from Neiva Colombia, we investigated the impact of two microclimate temperature profiles on ZIKV and CHIKV infection dynamics in Ae. aegypti. We found that the vector mortality was not significantly impacted by the difference in temperature profiles. Further, we found that the infection and dissemination rates were largely unaffected, with only ZIKV experiencing a significant increase in infection at outdoor temperatures at 21 days post-infection (dpi). Further, there was a significant increase in viral titers in the abdomens of ZIKV-infected mosquitoes at 21 dpi. With CHIKV, there was a significant titer difference in the abdomens of mosquitoes at both 7 and 14 dpi. While there were differences in vector infection kinetics that were not statistically significant, we developed a simple stochastic SEIR-SEI model to determine if the observed differences might translate to notable differences in simulated outbreaks. With ZIKV, while the probability of secondary transmission was high (&gt;90%) under both microenvironmental scenarios, there was often only one secondary case. However, CHIKV differences between microenvironments were more prominent. With over 90% probability of secondary transmission, at indoor conditions, the peak of transmission was higher (over 850 cases) compared to the outdoor conditions (&lt;350 cases). Further, the time-to-peak for indoor was 130 days compared to 217 days for outdoor scenarios. Further investigations into microenvironmental conditions, including temperature, may be key to increasing our understanding of the nuances of CHIKV and ZIKV vectorial capacity, epidemiology, and risk assessment, especially as it affects other aspects of transmission, such as biting rate. Overall, it is critical to understand the variability of how extrinsic factors affect transmission systems, and these data add to the growing catalog of knowledge of how temperature affects arboviral systems.

Microbiota Composition Associates With Mosquito Productivity Outcomes in Belowground Larval Habitats.

Vector mosquitoes are well-adapted to habitats in urban areas, including belowground infrastructure such as stormwater systems. As a major source of larval habitat in population centers, control of larval populations in stormwater catch basins is an important tool for control of vector-borne disease. Larval development and adult phenotypes driving vectorial capacity in mosquitoes are modulated by the larval gut microbiota, which is recruited from the aquatic environment in which larvae develop. Laboratory studies have quantified microbe-mediated impacts on individual mosquito phenotypes, but more work is needed to characterise how microbiota variation shapes population-level outcomes. Here, we evaluated the relationship between habitat microbiota variation and mosquito population dynamics by simultaneously characterising microbiota diversity, water quality, and mosquito productivity in a network of stormwater catch basins in the Chicago metropolitan area. High throughput sequencing of 16S rRNA gene amplicons from water samples collected from 60 basins over an entire mosquito breeding season detected highly diverse bacterial communities that varied with measures of water quality and over time. Insitu measurements of mosquito abundance in the same basins further varied by microbiota composition and the relative abundance of specific bacterial taxa. Altogether, these results illustrate the importance of habitat microbiota in shaping ecological processes that affect mosquito populations. They also lay the foundation for future studies to characterise the mechanisms by which specific bacterial taxa impact individual and population-level phenotypes related to mosquito vectorial capacity.

Automated identification of Chagas disease vectors using AlexNet pre-trained convolutional neural networks.

The 158 bug species that make up the subfamily Triatominae are the potential vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Despite recent progress in developing a picture-based automated system for identification of triatomines, an extensive and diverse image database is required for a broadly useful automated application for identifying these vectors. We evaluated performance of a deep-learning network (AlexNet) for identifying triatomine species from a database of dorsal images of adult insects. We used a sample of photos of 6397 triatomines belonging to seven genera and 65 species from 27 countries. AlexNet had an accuracy of ~0.93 (95% confidence interval [CI], 0.91-0.94) for identifying triatomine species from pictures of varying resolutions. Highest specific accuracy was observed for 21 species in the genera Rhodnius and Panstrongylus. AlexNet performance improved to ~0.95 (95% CI, 0.93-0.96) when only the species with highest vectorial capacity were considered. These results show that AlexNet, when trained with a large, diverse, and well-structured picture set, exhibits excellent performance for identifying triatomine species. This study contributed to the development of an automated Chagas disease vector identification system.

Alphaviral backbone of self-amplifying RNA enhances protein expression and immunogenicity against SARS-CoV-2 antigen.

Self-amplifying RNA (saRNA) vectors are a next-generation RNA technology that extends the expression of heterologous genes. Clinical trials have shown the dose-sparing capacity of saRNA vectors in a vaccine context compared with conventional messenger RNA. However, saRNA vectors have historically been based on a limited number of alphaviruses, and only the Venezuelan equine encephalitis virus-based saRNA vaccines have been used clinically. Here, we designed genotypically distinct alphaviral saRNA vectors and characterized their performance in mammalian cell lines, human skin explants and mice. Five of the 12 vectors had substantial luciferase expression in mice with variable pharmacokinetics, enabling modulation of both the magnitude and duration of protein expression. Additionally, we demonstrated that the alphaviral genotype of the saRNA significantly impacts the immunogenicity of saRNA vaccines, including the humoral and cellular responses in mice. Given the differences in RNA reactogenicity and expression between mice and humans, we assessed the saRNA vectors in human skin explants obtained from patients and observed high transgene expression. saRNA bioluminescence and immunogenicity in different mice strains were highly correlative, while minimal correlation was observed when compared with human explants and mammalian cell lines. This work demonstrates that efficacious saRNA vaccines and therapies can be produced by adapting genetically diverse alphaviruses into vectors.

A convenient viral transduction based method for advanced multi-engineering of primary human (CAR) T-cells

The past decades have illustrated the power of T-cell engineering in the development of new and successful cell therapies, such as chimeric antigen receptor (CAR) T-cells. Despite clinical success in hematological malignancies, it also becomes increasingly clear that additional T-cell engineering will be required to improve efficacy and safety and expand the application to solid tumors. Engineering is most often achieved by viral delivery of transgenes, however, viral vector capacity limitations make efficient and reproducible generation of multi transgene expressing T-cell therapeutics technically challenging. We here describe a convenient and efficient method for the delivery of up to three γ-retroviral CAR vectors in T-cells. We achieved this using virus vector mixtures that are simultaneously produced at high titers by double- or triple- transduced stable virus producer cells. We show that this method is superior in overall efficiency and reproducibility to conventional double or triple CAR transductions, in which separate viral batches are used. Due to its robustness, this method can facilitate the research and the development for advanced T-cell engineering towards more effective and safe therapies.

Microbiota composition of Culex perexiguus mosquitoes during the West Nile virus outbreak in southern Spain.

West Nile virus (WNV) is a flavivirus naturally circulating between mosquito vectors and birds, occasionally infecting horses and humans and causing epidemiologically relevant outbreaks. In Spain, the first big WNV outbreak was recorded in 2020, resulting in 77 people infected and 8 fatalities, most of them in southern Spain. Culex perexiguus was identified as the primary vector of WNV maintaining its enzootic circulation of the virus. Growing evidence highlights the role of mosquito microbiota as a key component determining the vectorial capacity of mosquitoes, largely contributing to disease epidemiology. Here, we develop, to our knowledge, the first identification of the microbiota composition of this mosquito vector under natural conditions and test for the potential relationship between mosquito microbiota composition and WNV infection. To do so, we collected mosquitoes in a natural area of southern Spain during the 2020 WNV outbreak and identified the microbiota composition of mosquitoes using a 16S rRNA gene metabarcoding approach. The microbiota of Cx. perexiguus was dominated by the phylum Proteobacteria. The most abundant families were Burkholderiaceae and Erwiniaceae, including the genera Burkholderia, Erwinia, and Pantoea. The genus Wolbachia, which use to dominate the microbiota of Cx. pipiens and negatively interact with WNV according to the literature, had a low prevalence and relative abundance in Cx. perexiguus and its abundance did not differ between WNV-positive and WNV-negative mosquito pools. The microbiota diversity and composition of Cx. perexiguus were not significantly related to the WNV infection status. These results provide the first identification of the mosquito microbiota in an endemic area of WNV circulation in Spain.

Vector-borne disease outbreak control via instant releases

This paper is devoted to the study of optimal release strategies to control vector-borne diseases, such as dengue, Zika, chikungunya and malaria. Two techniques are considered: the sterile insect one (SIT), which consists in releasing sterilized males among wild vectors in order to perturb their reproduction, and the Wolbachia one (presently used mainly for mosquitoes), which consists in releasing vectors, that are infected with a bacterium limiting their vectorial capacity, in order to replace the wild population by one with reduced vectorial capacity. In each case, the time dynamics of the vector population is modeled by a system of ordinary differential equations in which the releases are represented by linear combinations of Dirac measures with positive coefficients determining their intensity. We introduce optimal control problems that we solve numerically using ad-hoc algorithms, based on writing first-order optimality conditions characterizing the best combination of Dirac measures. We then discuss the results obtained, focusing in particular on the complexity and efficiency of optimal controls and comparing the strategies obtained. Mathematical modeling can help testing a great number of scenarios that are potentially interesting in future interventions (even those that are orthogonal to the present strategies) but that would be hard, costly or even impossible to test in the field in present conditions.

Mammalian ubiquitous promoter isolated from proximal regulatory region of bovine MSTN gene

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy.