Tips and tools to obtain and assess mosquito viromes.

Due to their vectorial capacity, mosquitoes (Diptera: Culicidae) receive special attention from health authorities and entomologists. These cosmopolitan insects are responsible for the transmission of many viral diseases, such as dengue and yellow fever, causing huge impacts on human health and justifying the intensification of research focused on mosquito-borne diseases. In this context, the study of the virome of mosquitoes can contribute to anticipate the emergence and/or the reemergence of infectious diseases. The assessment of mosquito viromes also contributes to the surveillance of a wide variety of viruses found in these insects, allowing the early detection of pathogens with public health importance. However, the study of mosquito viromes can be challenging due to the number and complexities of steps involved in this type of research. Therefore, this article aims to describe, in a straightforward and simplified way, the steps necessary for obtention and assessment of mosquito viromes. In brief, this article explores: the capture and preservation of specimens; sampling strategies; treatment of samples before DNA/RNA extraction; extraction methodologies; enrichment and purification processes; sequencing choices; and bioinformatics analysis.

Predictive modelling of Ross River virus using climate data in the Darling Downs.

Ross River virus (RRV) is the most common mosquito-borne infection in Australia. RRV disease is characterised by joint pain and lethargy, placing a substantial burden on individual patients, the healthcare system and economy. This burden is compounded by a lack of effective treatment or vaccine for the disease. The complex RRV disease ecology cycle includes a number of reservoirs and vectors that inhabit a range of environments and climates across Australia. Climate is known to influence humans, animals and the environment and has previously been shown to be useful to RRV prediction models. We developed a negative binomial regression model to predict monthly RRV case numbers and outbreaks in the Darling Downs region of Queensland, Australia. Human RRV notifications and climate data for the period July 2001 - June 2014 were used for model training. Model predictions were tested using data for July 2014 - June 2019. The final model was moderately effective at predicting RRV case numbers (Pearson's r = 0.427) and RRV outbreaks (accuracy = 65%, sensitivity = 59%, specificity = 73%). Our findings show that readily available climate data can provide timely prediction of RRV outbreaks.

Basic reproduction ratio of a mosquito-borne disease in heterogeneous environment.

To explore the influence of spatial heterogeneity on mosquito-borne diseases, we formulate a reaction-diffusion model with general incidence rates. The basic reproduction ratio [Formula: see text] for this model is introduced and the threshold dynamics in terms of [Formula: see text] are obtained. In the case where the model is spatially homogeneous, the global asymptotic stability of the endemic equilibrium is proved when [Formula: see text]. Under appropriate conditions, we establish the asymptotic profiles of [Formula: see text] in the case of small or large diffusion rates, and investigate the monotonicity of [Formula: see text] with respect to the heterogeneous diffusion coefficients. Numerically, the proposed model is applied to study the dengue fever transmission. Via performing simulations on the impacts of certain factors on [Formula: see text] and disease dynamics, we find some novel and interesting phenomena which can provide valuable information for the targeted implementation of disease control measures.

Ecological and clinical evidence of the establishment of West Nile virus in a large urban area in Europe, Berlin, Germany, 2021 to 2022.

BackgroundWest Nile virus (WNV), found in Berlin in birds since 2018 and humans since 2019, is a mosquito-borne virus that can manifest in humans as West Nile fever (WNF) or neuroinvasive disease (WNND). However, human WNV infections and associated disease are likely underdiagnosed.AimWe aimed to identify and genetically characterise WNV infections in humans and mosquitoes in Berlin.MethodsWe investigated acute WNV infection cases reported to the State Office for Health and Social Affairs Berlin in 2021 and analysed cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown aetiology (n = 489) for the presence of WNV. Mosquitoes were trapped at identified potential exposure sites of cases and examined for WNV infection.ResultsWest Nile virus was isolated and sequenced from a blood donor with WNF, a symptomatic patient with WNND and a WNND case retrospectively identified from testing CSF. All cases occurred in 2021 and had no history of travel 14 days prior to symptom onset (incubation period of the disease). We detected WNV in Culex pipiens mosquitoes sampled at the exposure site of one case in 2021, and in 2022. Genome analyses revealed a monophyletic Berlin-specific virus clade in which two enzootic mosquito-associated variants can be delineated based on tree topology and presence of single nucleotide variants. Both variants have highly identical counterparts in human cases indicating local acquisition of infection.ConclusionOur study provides evidence that autochthonous WNV lineage 2 infections occurred in Berlin and the virus has established an endemic maintenance cycle.

Eastern, Western, and Venezuelan Equine Encephalitis and West Nile Viruses: Clinical and Public Health Considerations.

The continued recognition and emergence of alphavirus and flavivirus diseases is a growing veterinary and public health concern. As the global environment continues to change, mosquito-borne diseases will continue to evolve and expand. Continued development of readily available vaccines for the prevention of these diseases in humans and animals is essential to controlling epizootics of these diseases. Further research into effective antiviral treatments is also sorely needed. This article describes equine encephalitis viruses with a focus on clinical and public health considerations.

West Nile virus in Europe: after action reviews of preparedness and response to the 2018 transmission season in Italy, Slovenia, Serbia and Greece.

BACKGROUND: After Action Reviews (AAR) with a One Health perspective were performed in Slovenia, Italy, Serbia and Greece following a severe West Nile virus (WNV) transmission season in 2018. A protocol combining traditional techniques and organizational process analysis was developed and then implemented in each country. RESULTS: In 2018, response to the unusually intense transmission season of WNV in Slovenia, Italy, Serbia and Greece took place through routine response mechanisms. None of the four countries declared a national or subnational emergency. We found a very strong consensus on the strengths identified in responding to this event. All countries indicated the availability of One Health Plans for surveillance and response; very high laboratory diagnostic capacity in the human, veterinary and entomology sectors and strong inter-sectoral collaboration with strong commitment of engaged institutions as critical in the management of the event. Finally, countries implementing One Health surveillance for WNV (in terms of early warning and early activation of prevention measures) consistently reported a positive impact on their activities, in particular when combining mosquito and bird surveillance with surveillance of cases in humans and equids. Recurring priority areas for improvement included: increasing knowledge on vector-control measures, ensuring the sustainability of vector monitoring and surveillance, and improving capacity to manage media pressure. CONCLUSIONS: The AARs presented here demonstrate the benefit of cross-sectoral and cross-disciplinary approaches to preparedness for West Nile virus outbreaks in Europe. In the coming years, priorities include fostering and strengthening arrangements that: enable coordinated One Health surveillance and response during WNV transmission seasons; ensure adequate laboratory capacities; strengthen risk communication; and fund longer-term research to address the knowledge gaps identified in this study.

A Population Dynamics Model of Mosquito-Borne Disease Transmission, Focusing on Mosquitoes' Biased Distribution and Mosquito Repellent Use.

We present an improved mathematical model of population dynamics of mosquito-borne disease transmission. Our model considers the effect of mosquito repellent use and the mosquito's behavior or attraction to the infected human, which cause mosquitoes' biased distribution around the human population. Our analysis of the model clearly shows the existence of thresholds for mosquito repellent efficacy and its utilization rate in the human population with respect to the elimination of mosquito-borne diseases. Further, the results imply that the suppression of mosquito-borne diseases becomes more difficult when the mosquitoes' distribution is biased to a greater extent around the human population.

A genetic algorithm for identifying spatially-varying environmental drivers in a malaria time series model.

Time series models of malaria cases can be applied to forecast epidemics and support proactive interventions. Mosquito life history and parasite development are sensitive to environmental factors such as temperature and precipitation, and these variables are often used as predictors in malaria models. However, malaria-environment relationships can vary with ecological and social context. We used a genetic algorithm to optimize a spatiotemporal malaria model by aggregating locations into clusters with similar environmental sensitivities. We tested the algorithm in the Amhara Region of Ethiopia using seven years of weekly Plasmodium falciparum data from 47 districts and remotely-sensed land surface temperature, precipitation, and spectral indices as predictors. The best model identified six clusters, and the districts in each cluster had distinctive responses to the environmental predictors. We conclude that spatial stratification can improve the fit of environmentally-driven disease models, and genetic algorithms provide a practical and effective approach for identifying these clusters.

Evaluation of an immunochromatography rapid diagnosis kit for detection of chikungunya virus antigen in India, a dengue-endemic country.

BACKGROUND: Chikungunya virus (CHIKV) and dengue virus (DENV) are arboviruses that share the same Aedes mosquito vector, and there is much overlap in endemic areas. In India, co-infection with both viruses is often reported. Clinical manifestations of Chikungunya fever is often confused with dengue fever because clinical symptoms of both infections are similar. It is, therefore, difficult to differentiate from those of other febrile illnesses, especially dengue fever. We previously developed a CHIKV antigen detection immunochromatography (IC) rapid diagnosis kit [1]. The current study examined the efficacy of previously mentioned IC kit in India, a dengue-endemic country. METHODS: Sera from 104 CHIKV-positive (by qRT-PCR) and/or IgM-positive (ELISA) subjects collected in 2016, were examined. Fifteen samples from individuals with CHIKV-negative/DENV-positive and 4 samples from healthy individuals were also examined. Of the 104 CHIKV-positive sera, 20 were co-infected with DENV. RESULTS: The sensitivity, specificity and overall agreement of the IC assay were 93.7, 95.5 and 94.3%, respectively, using qRT-PCR as a gold standard. Also, there was a strong, statistically significant positive correlation between the IC kit device score and the CHIKV RNA copy number. The IC kit detected CHIKV antigen even in DENV-co-infected patient sera and did not cross-react with DENV NS1-positive/CHIKV-negative samples. CONCLUSIONS: The results suggest that the IC kit is useful for rapid diagnosis of CHIKV in endemic areas in which both CHIKV and DENV are circulating.

Fine-temporal forecasting of outbreak probability and severity: Ross River virus in Western Australia.

Health warnings of mosquito-borne disease risk require forecasts that are accurate at fine-temporal resolutions (weekly scales); however, most forecasting is coarse (monthly). We use environmental and Ross River virus (RRV) surveillance to predict weekly outbreak probabilities and incidence spanning tropical, semi-arid, and Mediterranean regions of Western Australia (1991-2014). Hurdle and linear models were used to predict outbreak probabilities and incidence respectively, using time-lagged environmental variables. Forecast accuracy was assessed by model fit and cross-validation. Residual RRV notification data were also examined against mitigation expenditure for one site, Mandurah 2007-2014. Models were predictive of RRV activity, except at one site (Capel). Minimum temperature was an important predictor of RRV outbreaks and incidence at all predicted sites. Precipitation was more likely to cause outbreaks and greater incidence among tropical and semi-arid sites. While variable, mitigation expenditure coincided positively with increased RRV incidence (r 2 = 0·21). Our research demonstrates capacity to accurately predict mosquito-borne disease outbreaks and incidence at fine-temporal resolutions. We apply our findings, developing a user-friendly tool enabling managers to easily adopt this research to forecast region-specific RRV outbreaks and incidence. Approaches here may be of value to fine-scale forecasting of RRV in other areas of Australia, and other mosquito-borne diseases.

Characterization and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies.

Mosquitoes (Diptera: Culicidae) serve as important vectors for a wide number of parasites and pathogens of huge medical and veterinary importance. Aedes aegypti is a primary dengue vector in tropical and subtropical urban areas. There is an urgent need to develop eco-friendly mosquitocides. In this study, silver nanoparticles (AgNP) were biosynthesized using neem cake, a by-product of the neem oil extraction from the seed kernels of Azadirachta indica. AgNP were characterized using a variety of biophysical methods, including UV-vis spectrophotometry, FTIR, SEM, EDX, and XRD analyses. Furthermore, the neem cake extract and the biosynthesized AgNP were tested for acute toxicity against larvae and pupae of the dengue vector Ae. aegypti. LC50 values achieved by the neem cake extract ranged from 106.53 (larva I) to 235.36 ppm (pupa), while AgNP LC50 ranged from 3.969 (larva I) to 8.308 ppm (pupa). In standard laboratory conditions, the predation efficiency of a Carassius auratus per day was 7.9 (larva II) and 5.5 individuals (larva III). Post-treatment with sub-lethal doses of AgNP, the predation efficiency was boosted to 9.2 (larva II) and 8.1 individuals (larva III). The genotoxic effect of AgNP was studied on C. auratus using the comet assay and micronucleus frequency test. DNA damage was evaluated on peripheral erythrocytes sampled at different time intervals from the treatment; experiments showed no significant damages at doses below 12 ppm. Overall, this research pointed out that neem cake-fabricated AgNP are easy to produce, stable over time, and can be employed at low dosages to reduce populations of dengue vectors, with moderate detrimental effects on non-target mosquito natural enemies.

Epidemiological Characteristics and Risk Factors of Dengue Infection in Korean Travelers.

Dengue viral infection has rapidly spread around the world in recent decades. In Korea, autochthonous cases of dengue fever have not been confirmed yet. However, imported dengue cases have been increased since 2001. The risk of developing severe dengue in Korean has been increased by the accumulation of past-infected persons with residual antibodies to dengue virus and the remarkable growth of traveling to endemic countries in Southeast Asia. Notably, most of imported dengue cases were identified from July to December, suggesting that traveling during rainy season of Southeast Asia is considered a risk factor for dengue infection. Analyzing national surveillance data from 2011 to 2015, males aged 20-29 years are considered as the highest risk group. But considering the age and gender distribution of travelers, age groups 10-49 except 20-29 years old males have similar risks for infection. To minimize a risk of dengue fever and severe dengue, travelers should consider regional and seasonal dengue situation. It is recommended to prevent from mosquito bites or to abstain from repetitive visit to endemic countries. In addition, more active surveillance system and monitoring the prevalence asymptomatic infection and virus serotypes are required to prevent severe dengue and indigenous dengue outbreak.

A review on Zika vaccine development.

Zika virus (ZIKV), which belongs to the Flavivirus family, is mainly transmitted via the bite of Aedes mosquitoes. In newborns, ZIKV infection can cause severe symptoms such as microcephaly, while in adults, it can lead to Guillain‒Barré syndrome (GBS). Due to the lack of specific therapeutic methods against ZIKV, the development of a safe and effective vaccine is extremely important. Several potential ZIKV vaccines, such as live attenuated, inactivated, nucleic acid, viral vector, and recombinant subunit vaccines, have demonstrated promising outcomes in clinical trials involving human participants. Therefore, in this review, the recent developmental progress, advantages and disadvantages of these five vaccine types are examined, and practical recommendations for future development are provided.

Peptide-Based Flavivirus Biosensors: From Cell Structure to Virological and Serological Detection Methods.

In tropical and developing countries, mosquito-borne diseases by flaviviruses pose a serious threat to public health. Early detection is critical for preventing their spread, but conventional methods are time-consuming and require skilled technicians. Biosensors have been developed to address this issue, but cross-reactivity with other flaviviruses remains a challenge. Peptides are essentially biomaterials used in diagnostics that allow virological and serological techniques to identify flavivirus selectively. This biomaterial originated as a small protein consisting of two to 50 amino acid chains. They offer flexibility in chemical modification and can be easily synthesized and applied to living cells in the engineering process. Peptides could potentially be developed as robust, low-cost, sensitive, and selective receptors for detecting flaviviruses. However, modification and selection of the receptor agents are crucial to determine the effectiveness of binding between the targets and the receptors. This paper addresses two potential peptide nucleic acids (PNAs) and affinity peptides that can detect flavivirus from another target-based biosensor as well as the potential peptide behaviors of flaviviruses. The PNAs detect flaviviruses based on the nucleotide base sequence of the target's virological profile via Watson-Crick base pairing, while the affinity peptides sense the epitope or immunological profile of the targets. Recent developments in the functionalization of peptides for flavivirus biosensors are explored in this Review by division into electrochemical, optical, and other detection methods.

[How do female mosquitoes determine the most suitable males for mating?]

More than 80% of the world's populations are at risk of vector-borne diseases, with mosquito-borne diseases as a significant global public health problem. Mosquito populations control is critical to interrupting the transmission of mosquito-borne diseases. This review summarizes the physical attributes, smell, vision, touch, and hearing of mosquitoes to unravel the preferences of female mosquitoes, and describes the mechanisms underlying the best male mating by female mosquitoes, so as to provide new insights into management of mosquito-borne diseases.

Update on the composition and distribution of the mosquito fauna (Diptera: Culicidae) in Cabo Verde, a country at risk for mosquito-borne diseases.

Mosquitoes play a critical role as vectors of pathogens affecting both humans and animals. Therefore, understanding their biodiversity and distribution is crucial to developing evidence-based vector control strategies. The current study updated the composition and distribution of mosquito species through a comprehensive survey of all municipalities of Cabo Verde. From October 2017 to September 2018, mosquito larvae and pupae were collected from 814 aquatic habitats. Anopheles gambiae (Giles, 1902) and Culex pipiens (Linnaeus, 1758) complexes were subjected to PCR-based techniques for sibling species identification. Ten mosquito species from 5 genera were identified: Aedes aegypti (Linnaeus, 1762), Aedes caspius (Pallas, 1771), Anopheles arabiensis (Patton, 1905), Anopheles pretoriensis (Theobald, 1903), Culex bitaeniorhynchus (Giles, 1901), Cx. pipiens, Culex quinquefasciatus (Say, 1823), Culex tritaeniorhynchus (Giles, 1901), Culiseta longiareolata (Macquart, 1838), and Lutzia tigripes (de Grandpre & de Charmoy, 1901). Santiago Island reported the highest number of species (n = 8). Ae. aegypti and Cx. quinquefasciatus were the most widely distributed species across the country. An. arabiensis was the sole species identified within the An. gambiae complex. The findings from our study will help guide health policy decisions to effectively control mosquito-borne diseases.

West Nile Virus Infection in Occupational Settings-A Systematic Review.

BACKGROUND: West Nile virus (WNV) is an emerging mosquito-borne neurotropic virus, belonging to the Flaviviridae family and the Orthoflavivirus genus. The effective control of WNV requires a targeted preventive strategy that also needs the identification of the higher-risk populations. Hence, this study focused on a systematic literature review of WNV-acquired infection in work-related settings and the assessment of the exposure risks among different occupational categories. METHODS: A comprehensive search was conducted to identify studies until September 2023 in multiple databases such as PubMed/MEDLINE, SCOPUS and Web of Science, according to the PRISMA 2020 statement. Risk of bias of collected papers was assessed by the ROB tool of the National Toxicology Program's Office of Health Assessment and Translation handbook. RESULTS: A total of 21 studies were included in the systematic review, out of which seventeen were observational studies and four were case reports. Workers identified as at higher risk for WNV infection were military workers, veterinarians, agricultural workers, farmers, and laboratory workers with contact with infected fluids or aerosols. CONCLUSIONS: The identification of higher-risk workers could facilitate active surveillance by occupational physicians, which could improve our understanding of the epidemiology of WNV and, in addition, could help tailor appropriate preventive recommendations, reducing the overall burden of disease in high-risk areas.

The Distribution, Diversity, and Control of Dirofilariosis in Brazil: A Comprehensive Review.

Brazil's extensive coastline, tropical and subtropical climate, and well-preserved environment represent a conducive setting for dirofilariosis, a zoonotic mosquito-borne disease. Although this condition has long been recognised in the country, it has been relatively under-studied, and it is currently considered to be an emerging disease. Diagnosis, treatment, and control remain challenging due to the extensive gaps in knowledge. In order to help address this issue, this review aims to (i) summarise the available literature on the distribution of Dirofilaria spp. in Brazilian dogs over the last decade, (ii) review case reports of dirofilariosis in cats, wild animals, and humans over the last twenty years, and (iii) highlight the benefits of taking a One Health approach to managing this disease. While there have been several prevalence studies in dogs, disease distribution is poorly characterised in cats, and little is known about the occurrence of the parasite in wildlife. Human cases are sporadically reported, and no large-scale studies have been undertaken to date. Evidence indicates that Dirofilaria immitis is the main species circulating in Brazil, although Dirofilaria repens has also been detected. Molecular studies have also suggested the circulation of a highly virulent form of D. immitis, which may be genetically distinct from those of the Old World. A programme of epidemiological, ecological, genomic, and pathogenicity-based studies is required to quantify the impact of dirofilariosis in Brazil on both veterinary and public health and to inform others on its control.

Evolutionary adaptation under climate change: Aedes sp. demonstrates potential to adapt to warming.

Climate warming is expected to shift the distributions of mosquitoes and mosquito-borne diseases, facilitating expansions at cool range edges and contractions at warm range edges. However, whether mosquito populations could maintain their warm edges through evolutionary adaptation remains unknown. Here, we investigate the potential for thermal adaptation in Aedes sierrensis, a congener of the major disease vector species that experiences large thermal gradients in its native range, by assaying tolerance to prolonged and acute heat exposure, and its genetic basis in a diverse, field-derived population. We found pervasive evidence of heritable genetic variation in acute heat tolerance, which phenotypically trades off with tolerance to prolonged heat exposure. A simple evolutionary model based on our data shows that the estimated maximum rate of evolutionary adaptation in mosquito heat tolerance typically exceeds that of projected climate warming under idealized conditions. Our findings indicate that natural mosquito populations may have the potential to track projected warming via genetic adaptation. Prior climate-based projections may thus underestimate the range of mosquito and mosquito-borne disease distributions under future climate conditions.

The first confirmed outbreak of chikungunya reported in Timor-Leste, 2024.

Abstract: Timor-Leste is a mountainous, half-island nation with a population of 1.3 million, which shares a land border with Indonesia and is 550 km from Darwin, Australia. Since independence in 2002, Timor-Leste has achieved significant development; however, high levels of poverty remain. Chikungunya virus (CHIKV) is endemic in over 100 countries in Africa, Asia, Europe and in the Americas. It is transmitted by the bite of infected Aedes aegypti or Ae. albopictus mosquitoes, which are present in Timor-Leste and which contribute to annual rainy-season dengue virus (DENV) outbreaks. Symptomatic people typically suffer from acute onset of fever, usually accompanied by severe arthritis or arthralgia. Joint pain can be debilitating for several days, and may sometimes last for weeks, months or years. Unlike DENV infection which has significant mortality, most people recover completely. Between 2002 and 2023, there were 26 cases of CHIKV notified in Australia who acquired their infection in Timor-Leste; however, laboratory testing capability for CHIKV in Timor-Leste only became available in 2021 using polymerase chain reaction (PCR). The first locally diagnosed case was notified in November 2023. In January 2024, an outbreak of CHIKV was recognised in Timor-Leste for the first time, with 195 outbreak cases reported during 1-31 January 2024; all were PCR positive. There were no cases hospitalised, and no deaths. The median age of cases was 17 years (range 1-76 years); 51% were males. Cases were reported across the country; most (88/195) were from Dili, although the highest incidence was seen in the neighbouring municipality of Ermera (monthly incidence rate of 58.8 cases per 100,000 population). This first reported outbreak of CHIKV in Timor-Leste highlights the need for improved mosquito-borne illness control and response strategies, including minimising breeding sites and promoting early presentation for treatment and differential diagnosis from DENV, and consideration of the deployment of Wolbachia-infected mosquitoes, particularly as they have shown to reduce the transmission of CHIKV, DENV and Zika virus, all of which pose threats in Timor-Leste.