Using Rapid Analyte Measurement Platform (RAMP) as a Tool for an Early Warning System Assessing West Nile Virus Epidemiological Risk in Bucharest, Romania.

West Nile virus (WNV) is the most widely spread arbovirus in the world. Early detection of this virus in mosquito populations is essential for implementing rapid vector control measures to prevent outbreaks. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is a powerful tool for the detection of WNV in mosquito pools, but it is a time- and resource-consuming assay. We used a Rapid Analyte Measurement Platform (RAMP) assay in a vector surveillance program for rapid detection of WNV in mosquitoes collected in Bucharest city, Romania, in 2021. The positive mosquito pools were tested for confirmation with real-time RT-PCR. Three out of the 24 RAMP assay positive pools were not confirmed by real-time RT-PCR. We consider that RAMP assay can be used as a fast and reliable method for the screening of WNV presence in mosquito pools, but we recommend that samples with values ranging from 30 to 100 RAMP units should fall in a grey zone and should be considered for real-time RT-PCR confirmation.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Practical management plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus).

Aedes albopictus, also known as the "Asian Tiger Mosquito", is an invasive mosquito species to Europe causing high concern in public health due to its severe nuisance and its vectorial capacity for pathogens such as dengue, chikungunya, yellow fever and Zika. Consequently, the responsible authorities implement management activities to reduce its population density, possibly to below noxious and epidemiological thresholds. In urban areas, these aims are difficult to achieve because of the species' ability to develop in a wide range of artificial breeding sites, mainly private properties. This document (Management Plan) has been structured to serve as a comprehensive practical and technical guide for stakeholders in organizing the vector control activities in the best possible way. The current plan includes coordinated actions such as standardized control measures and quality control activities, monitoring protocols, activities for stakeholders and local communities, and an emergency vector control plan to reduce the risk of an epidemic.

The invasive Asian tiger mosquito Aedes albopictus in Romania: towards a country-wide colonization?

Currently, five invasive Aedes mosquito species are of concern in Europe according to the European Centre for Disease Prevention and Control: Aedes aegypti, Ae. albopictus, Ae. atropalpus, Ae. japonicus, and Ae. koreicus. Among these, only Ae. albopictus was reported to occur in Romania, in Bucharest. The aim of this study was to update the knowledge on the distribution of this invasive mosquito species in Romania, by investigating new potential locations. Monitoring of Ae. albopictus was carried out between May 2017 and October 2018. Three types of traps (CDC-Gravid Traps, CDC miniature Light Traps, ovitraps) were placed in 53 localities in 13 counties at sites suitable for container-breeding mosquitoes. Collected adult mosquitoes were counted and identified according to morphological criteria. Larvae were found present in domestic containers and rain catch basins. Aedes albopictus adults and eggs were collected in 10 localities in eight counties across Romania. Our study confirms nine new localities and seven counties where Ae. albopictus became established in Romania, highlighting the need for surveillance to further assess the species' distribution and abundance, as well as the pathogen transmission risk related to that vector species.

Transmission Dynamics of the West Nile Virus in Mosquito Vector Populations under the Influence of Weather Factors in the Danube Delta, Romania.

Mosquitoes were collected in the Danube Delta during the active seasons of 2011-2013. For Culex spp. mosquitoes, the abundance was calculated. Culex pipiens (sensu lato), (s.l.) and Culex modestus pools were tested for the presence of West Nile virus (WNV) genome, and the maximum likelihood of the infection rate was established. Mean daily temperatures and precipitation were obtained for the closest meteorological station. A negative binominal model was used to evaluate linkages between the temperature/precipitation and mosquito population size. A zero-inflated negative binomial model was used to test the relationship between the temperature and the infection rate. A single complex model for infection rate prediction was also used. The linkages were calculated for lag 0 and for 10 days earlier (lag 1), 20 days earlier (lag 2), and 30 days earlier (lag 3). Significant positive linkages (P < 0.001) were detected between temperature and mosquito population size for lag 1, lag 2, and lag 3. The linkages between temperature and infection rates were positive and significant for lag 2 and lag 3. Negative significant (P < 0.001) results were detected between precipitation and infection rates for lags 1, 2, and 3. The complex model showed that the best predictors for infection rate are the temperature, 20 days earlier (positive linkage) and the precipitation, 30 days earlier (negative linkage). Positive temperature anomalies in spring and summer and rainfall decrease contributed to the increase in the Culex spp. abundance and accelerated the WNV amplification in mosquito vector populations in the following weeks.

Established Population of the Invasive Mosquito Species Aedes albopictus in Romania, 2012-14.

During an entomological investigation carried out in Bucharest and surroundings in fall of 2012, 45 adult mosquitoes (38 females and 7 males) of Aedes albopictus were collected in a neighborhood from the southern area of the city. The morphological identification of the species was further confirmed by sequencing 2 mitochondrial DNA markers: the cytochrome c oxidase subunit I and NADH dehydrogenase subunit 5 genes. Aedes albopictus was collected again in 2013 in the same area from July until October. During late summer the species was found also in another location in the city, downtown Bucharest. Larvae were found in water barrels and other types of household containers, as well as in rain catch basins. In 2014, following a nuisance complaint of a Bucharest inhabitant, the entomological investigation found aggressive Ae. albopictus adults on his property that harbored many mosquito larvae in container-type breeding habitats. These findings are the 1st records of this invasive species and of its breeding population in Romania, and show maintenance of the species over 2 winter seasons. Surveillance of the species outside the area of the capital city was not performed, therefore it is not known whether Ae. albopictus has been introduced in other regions of the country. The presence of Ae. albopictus has been reported every year (2012-14) to competent public health authorities, stressing on the importance of surveillance and of implementation of control measures.

Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe.

BACKGROUND: There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission. METHODS: Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks. RESULTS: Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004