Insect-specific flaviviruses in Aedes mosquitoes in Greece.

Mosquitoes of the genus Aedes are known vectors of pathogenic flaviviruses, and insect-specific flaviviruses (ISFs) have been detected in members of this genus in numerous parts of the world. In order to gain insight into whether Aedes mosquitoes in Greece are infected by flaviviruses, 1173 Aedes spp. mosquitoes collected in 2010 and 2012 were grouped in 53 pools and tested by RT nested PCR using flavivirus generic primers. Eight pools (15.09 %) were found to be PCR positive: five pools (5/53, 9.4 %) contained RNA sequences related to Ochlerotatus caspius flavivirus (OCFV), an ISF previously detected in the Iberian peninsula, two pools (2/53, 3.8 %) contained sequences related to a mosquito flavivirus detected in Aedes vexans (AeveV) in Italy and the Czech Republic, and one pool contained a DNA sequence that was too short to identify accurately. The highest OCFV prevalence (12.9 %) was observed in August 2010 in the regional unit of Thessaloniki. Similar sequences were later obtained from two Culex spp. pools collected in 2013 in the same regions. A genetic difference of 0.2-1.4 % was seen among the Greek OCFV strains, which differed by 2.2-4.1 % from the Iberian strains and by 6.2-11.1 % from the Finnish Hanko virus. The genetic distances among strains varied depending on the genome region (genes for E, NS3 and NS5 proteins), with NS3 being the most variable. The present study shows no evidence of infection of Aedes mosquitoes with known pathogenic flaviviruses, but it expands the geographic distribution of OCFV in the eastern Mediterranean area. Any implication of ISFs for public health (either directly or through interactions with other flaviviruses in the mosquitoes) remains to be elucidated.

West Nile virus in mosquitoes in Greece.

Epidemics of West Nile virus (WNV) occurred for two consecutive years in Greece (in 2010 and 2011). A total of 16,116 adult Culex pipiens mosquitoes collected in two peripheries, Central Macedonia and Thessaly, were tested for WNV infection. WNV lineage 2 was detected in 6/296 mosquito pools, three in each year. The H249P substitution in the NS3 protein, previously associated with increased pathogenicity and thermotolerance, was detected in all six WNV-positive mosquito pools. When 21 individual C. pipiens mosquitoes were tested for the locus CQ11 to distinguish between the two C. pipiens forms, pipiens and molestus, 71.4% were identified as pipiens, 4.7% as molestus, and 19% as hybrid pipiens/molestus, giving the first evidence that both C. pipiens biotypes are present in Greece, with a significant proportion being hybrids. The exact role of the C. pipiens forms and hybrids in the WNV epidemiology, in combination or not with the H249P substitution in the virus genome, remains to be elucidated.

West Nile Virus in Culex Mosquitoes in Central Macedonia, Greece, 2022.

In 2022, Greece was the second most seriously affected European country in terms of the West Nile virus (WNV), after Italy. Specifically, Central Macedonia was the region with the most reported human cases (81.5%). In the present study, 30,816 female Culex pipiens sensu lato mosquitoes were collected from May to September 2022 in the seven regional units of Central Macedonia; they were then grouped into 690 pools and tested for WNV, while next-generation sequencing was applied to the samples, which showed a cycle threshold of Ct < 30 in a real-time RT-PCR test. WNV was detected in 5.9% of pools, with significant differences in the detection rate among regional units and months. It is of interest that in the Thessaloniki regional unit, where most of the human cases were observed, the virus circulation started earlier, peaked earlier, and lasted longer than in the other regional units. All sequences clustered into the Central European subclade of WNV lineage 2, and the virus strain differed from the initial Greek strain of 2010 by 0.52% and 0.27% at the nucleotide and amino acid levels, respectively. Signature substitutions were present, such as S73P and T157A in the prM and E structural proteins, respectively. The screening of mosquitoes provides useful information for virus circulation in a region with a potential for early warning, while the availability of whole-genome sequences is essential for further studies, including virus evolution.

Effects of climatic and environmental factors on mosquito population inferred from West Nile virus surveillance in Greece.

Mosquito-borne diseases' impact on human health is among the most prominent of all communicable diseases. With limited pool of tools to contrast these diseases, public health focus remains preventing mosquito-human contacts. Applying a hierarchical spatio-temporal Bayesian model on West Nile virus (WNV) surveillance data from Greece, we aimed to investigate the impact of climatic and environmental factors on Culex mosquitoes' population. Our spatio-temporal analysis confirmed climatic factors as major drivers of WNV-transmitting-Culex mosquitoes population dynamics, with temperature and long periods of moderate-to-warm climate having the strongest positive effect on mosquito abundance. Conversely, rainfall, high humidity, and wind showed a negative impact. The results suggest the presence of statistically significant differences in the effect of regional and seasonal characteristics, highlighting the complex interplay between climatic, geographical and environmental factors in the dynamics of mosquito populations. This study may represent a relevant tool to inform public health policymakers in planning preventive measures.

Detection and molecular characterization of West Nile virus in Culex pipiens mosquitoes in Central Macedonia, Greece, 2019-2021.

Since 2010 when West Nile virus (WNV) emerged in Greece, it causes seasonal outbreaks of human infections almost every year. During May-October of 2019-2021 a total number of 51,504 Culex pipiens mosquitoes were trapped in all seven regional units of Central Macedonia in northern Greece. They were grouped into 1099 pools and tested for WNV. The virus was detected in 5% of the mosquito pools (1.5%, 3.6% and 9.6% pools in 2019, 2020, and 2021, respectively), with significant rate differences among the regional units and years. The highest maximum likelihood estimation for WNV infection rates calculated per 1000 mosquitoes for 2019 and 2020 were 1.89 and 3.84 in Serres, and 7.08 for 2021 in Pella regional unit. Sixteen whole genome sequences were taken by applying a recently described PCR-based next generation sequencing protocol. Phylogenetic analysis showed that the sequences belonged to the Central European clade of WNV lineage 2, and that a virus strain introduced in Greece in 2019 continued to circulate and spread further during 2020-2021. The data are useful for public health and mosquito control programs' operational scheduling, while the whole genome sequences are an added value for molecular epidemiology and evolutionary studies.

Mosquito population structure, pathogen surveillance and insecticide resistance monitoring in urban regions of Crete, Greece.

BACKGROUND: In Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece's major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority. METHODOLOGY: Under the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018-2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species. PRINCIPAL FINDINGS: Important disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25-46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses. CONCLUSIONS/SIGNIFICANCE: The findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.

AIMSurv: First pan-European harmonized surveillance of Aedes invasive mosquito species of relevance for human vector-borne diseases.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named "AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108". AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

West Nile fever upsurge in a Greek regional unit, 2020.

During the 2020 West Nile virus (WNV) transmission season, Greece was the most affected EU Member State. More than one third of human cases occurred in Serres regional unit in northern Greece, which is characterized by the presence of a major wetland (Kerkini lake and Strimon river). A total of 2809 Culex pipiens mosquitoes collected in Serres were grouped into 70 pools and tested for WNV. Ten (14.3%) pools were found positive, and all WNV sequences belonged to the Central European subclade of WNV lineage 2. The first human case occurred in a village nearby the lake, and all following cases occurred across the connected river and its tributaries. Similar distribution presented the sites where WNV-positive mosquitoes were detected. The number of Culex spp. mosquitoes per trap per night was higher in 2020 than in previous years (2017-2019). The spatial and temporal distribution of human cases and WNV-positive mosquitoes in 2020 in Serres regional unit suggest that the upsurge of the virus circulation was probably related with factors that affected the ecosystem of the wetland.

West Nile virus lineage 2 in Culex mosquitoes in Thessaly, Greece, 2019.

West Nile virus is a flavivirus transmitted to humans mainly by mosquito bites. Outbreaks are observed in several European countries, and Greece is one of the most affected countries during the recent years. Thessaly was one of the most affected regions in Greece in 2019. A total of 3,025 Culex spp. mosquitoes collected in Thessaly were grouped into 47 pools and tested for West Nile virus (WNV). Eight (17%) pools were found positive. Whole genome sequences were obtained from two positive pools. Phylogenetic analysis showed that the causative strain was an evolutionary variant of the strains circulating in 2018 belonging to the Balkan subgroup of the Central European subclade of WNV lineage 2.

Detection of flaviviruses and alphaviruses in mosquitoes in Central Macedonia, Greece, 2018.

Culex mosquitoes are vectors of several flaviviruses and alphaviruses posing a potential risk to public and veterinary health. In order to gain an insight into the flaviviruses and alphaviruses circulating in the five regional units of Central Macedonia in northern Greece, 17,470 female Culex spp. mosquitoes collected during 2018 were tested for these viruses. Among 229 mosquito pools, West Nile virus (WNV) was detected in 10 (4.4%) pools, while insect-specific flavi- and alphaviruses were detected in 2 (0.9%) and 8 (3.5%) pools, respectively. WNV minimum infection rate (MIR) was 0.57. The highest MIR was identified in Thessaloniki regional unit, where several human cases of WNV infection occurred in 2018. All ten WNV sequences cluster into the Central European subclade of lineage 2. It is of note that the first WNV-positive mosquito pool was detected two weeks prior the report of the first human case in the area, suggesting that testing of mosquitoes could serve as early warning system.

Prediction of Antioxidant Activity of Cherry Fruits from UAS Multispectral Imagery Using Machine Learning.

In this research, a model for the estimation of antioxidant content in cherry fruits from multispectral imagery acquired from drones was developed, based on machine learning methods. For two consecutive cultivation years, the trees were sampled on different dates and then analysed for their fruits' radical scavenging activity (DPPH) and Folin-Ciocalteu (FCR) reducing capacity. Multispectral images from unmanned aerial vehicles were acquired on the same dates with fruit sampling. Soil samples were collected throughout the study fields at the end of the season. Topographic, hydrographic and weather data also were included in modelling. First-year data were used for model-fitting, whereas second-year data for testing. Spatial autocorrelation tests indicated unbiased sampling and, moreover, allowed restriction of modelling input parameters to a smaller group. The optimum model employs 24 input variables resulting in a 6.74 root mean square error. Provided that soil profiles and other ancillary data are known in advance of the cultivation season, capturing drone images in critical growth phases, together with contemporary weather data, can support site- and time-specific harvesting. It could also support site-specific treatments (precision farming) for improving fruit quality in the long-term, with analogous marketing perspectives.

Records of Aedes albopictus (Skuse, 1894) (Diptera; Culicidae) and Culex tritaeniorhynchus (Diptera; Culicidae) Expansion in Areas in Mainland Greece and Islands.

Annual entomological surveillance programs aiming to monitor mosquito populations and record presence and absence of mosquito species have been performed in Greece. We report, in this study, new records and expansion of Aedes albopictus in the islands of Lesvos (region of North Aegean), Crete (region of Crete), and the regional units of Rodopi in East Macedonia-Thrace. Furthermore, Culex tritaeniorhynchus was recorded for the first time in Arta (region of Epirus) in northwestern Greece.

Serological monitoring of backyard chickens in Central Macedonia-Greece can detect low transmission of West Nile virus in the absence of human neuroinvasive disease cases.

During 2010-13, West Nile virus (WNV) epidemics occurred in Greece with high numbers of human cases. In parallel, WNV serological surveillance utilizing domestic birds was applied mainly in Central Macedonia, as well as in other areas of the country, and allowed efficient detection of WNV activity during this period. The objective of the study was to evaluate the sensitivity of chicken-based WNV surveillance in periods of low-level virus transmission (2014-15) in a well-studied area, i.e. the epicenter of the 2010 WNV epidemic (Central Macedonia), which is considered endemic since then. WNV activity was monitored via determination of antiviral immune responses in juvenile backyard chickens. The birds were sampled twice per transmission period. WNV-specific antibodies were detected by ELISA in 2.8% out of 255 chickens sampled early in the 2014 transmission period (95% CI: 1-6%). Continued virus transmission was detected at the end of the period, as 4.2% out of 240 sampled chickens seroconverted to WNV (95% CI: 2-8%). Although 14 human neuroinvasive cases occurred in Greece during 2014, no such cases were reported in the study area. During the 2015 early warning period, antibodies against WNV were not detected in sampled chickens (n=250, 95% CI: 0-2%). However, humoral immune responses were detected in 6 out of 240 chicken sampled at the end of the transmission period (2.5%; 95% CI: 1-6%), indicating continued WNV activity. No human cases were reported in Greece during 2015. All samples were negative with real-time RT-PCR. Serological surveillance of chickens resulted in identification of areas with low WNV activity levels during 2014-15, and provided indications of its overwintering in Central Macedonia. The findings suggest that surveillance based on serological testing of domestic birds is sensitive and able to detect low-level of WNV enzootic transmission, in the absence of human cases.

Identification of Climatic Factors Affecting the Epidemiology of Human West Nile Virus Infections in Northern Greece.

Climate can affect the geographic and seasonal patterns of vector-borne disease incidence such as West Nile Virus (WNV) infections. We explore the association between climatic factors and the occurrence of West Nile fever (WNF) or West Nile neuro-invasive disease (WNND) in humans in Northern Greece over the years 2010-2014. Time series over a period of 30 years (1979-2008) of climatic data of air temperature, relative humidity, soil temperature, volumetric soil water content, wind speed, and precipitation representing average climate were obtained utilising the ECMWF's (European Centre for Medium-Range Weather Forecasts) Re-Analysis (ERA-Interim) system allowing for a homogeneous set of data in time and space. We analysed data of reported human cases of WNF/WNND and Culex mosquitoes in Northern Greece. Quantitative assessment resulted in identifying associations between the above climatic variables and reported human cases of WNF/WNND. A substantial fraction of the cases was linked to the upper percentiles of the distribution of air and soil temperature for the period 1979-2008 and the lower percentiles of relative humidity and soil water content. A statistically relevant relationship between the mean weekly value climatic anomalies of wind speed (negative association), relative humidity (negative association) and air temperature (positive association) over 30 years, and reported human cases of WNF/WNND during the period 2010-2014 could be shown. A negative association between the presence of WNV infected Culex mosquitoes and wind speed could be identified. The statistically significant associations could also be confirmed for the week the WNF/WNND human cases appear and when a time lag of up to three weeks was considered. Similar statistically significant associations were identified with the weekly anomalies of the maximum and minimum values of the above climatic factors. Utilising the ERA-Interim re-analysis methodology it could be shown that besides air temperature, climatic factors such as soil temperature, relative humidity, soil water content and wind speed may affect the epidemiology of WNV.

Detection of West Nile virus and insect-specific flavivirus RNA in Culex mosquitoes, central Macedonia, Greece.

BACKGROUND: West Nile virus (WNV) emerged in central Macedonia, northern Greece, in 2010. The aim of the study was to test Culex mosquitoes for genetic detection of WNV and other flaviviruses, and to check whether the detection of WNV in mosquitoes was correlated with the occurrence of human WNV cases. METHODS: During 2013, 25 780 Culex spp. mosquitoes were collected from central Macedonia, grouped into 295 pools, according to collection site and date, and tested for the presence of flavivirus RNA. RESULTS: Thirteen (4.4%) pools were flavivirus-positive. WNV lineage 2 was detected in nine (3.1%) pools, and insect-specific flaviviruses (ISFV) in four (1.4%). Three ISFV nucleotide sequences were 98% identical to Culex theileri flavivirus, previously detected in the Iberian peninsula, while the fourth sequence differed by >25% from all known flaviviruses. In most units of central Macedonia WNV detection in mosquitoes preceded the occurrence of human cases by approximately one month. CONCLUSIONS: Detection of WNV lineage 2 in mosquitoes for a fourth consecutive year suggests that the virus is established in Greece. Entomological surveillance provides an early warning system for the circulation of the virus. The detection of Culex theileri flavivirus extends the known range of this virus.

Evaluation of a West Nile virus surveillance and early warning system in Greece, based on domestic pigeons.

In the summer of 2010 an epidemic of West Nile virus (WNV) occurred in Central Macedonia, Greece, with 197 human neuroinvasive disease (WNND) cases. In the following years the virus spread to new areas, with a total of 76 WNND cases in 2011, and 109 WNND cases in 2012 (14 and 12 WNND cases, respectively, in Central Macedonia). We established a surveillance system based on serological testing of domestic pigeons, using cELISA confirmed by serum neutralization test. In Central Macedonia, pigeon seroprevalence was 54% (95% CI: 49-59%) and 31% (95% CI: 24-37%) at the end of the 2010 and 2011 epidemic seasons, respectively. One serum was positive for neutralizing antibodies directed against Usutu virus. Pigeon WNV seroprevalence and incidence rates of human WNND after the 2010 epidemic were positively correlated (ρ=0.94, at the regional unit level), while in 2011 the correlation (ρ=0.56) was not statistically significant, possibly due to small number of human WNND cases recorded. To evaluate the efficacy of the system at alerting upon WNV enzootic circulation before the onset of human cases, we tested 270 pigeons in 2011 and 240 pigeons in 2012. In Central Macedonia, the first seroconversions in pigeons were recorded 44 and 47 days, respectively, before the first human WNND cases. Pigeon surveillance was used successfully for identification of areas with WNV enzootic transmission and for early warning. Timely diffusion of information to health authorities facilitated the implementation of preparedness plans to protect public health.

Population seroprevalence study after a West Nile virus lineage 2 epidemic, Greece, 2010.

INTRODUCTION: During summer 2010, 262 human cases including 35 deaths from West Nile virus (WNV) infection were reported from Central Macedonia, Greece. Evidence from mosquitoes, birds and blood donors demonstrated that the epidemic was caused by WNV lineage 2, which until recently was considered of low virulence. We conducted a household seroprevalence study to estimate the spread of infection in the population during the epidemic, ascertain the relationship of infection to clinical disease, and identify risk factors for infection. METHODS: We used a two-stage cluster design to select a random sample of residents aged ≥18 years in the outbreak epicentre. We collected demographic, medical, and risk factor data using standard questionnaires and environmental checklists, and tested serum samples for presence of WNV IgG and IgM antibodies using ELISA. RESULTS: Overall, 723 individuals participated in the study, and 644 blood samples were available. Weighted seropositivity for IgG antibodies was 5.8% (95% CI: 3.8-8.6; n=41). We estimated that about 1 in 130 (1:141 to 1:124) infected individuals developed WNV neuroinvasive disease, and approximately 18% had clinical manifestations attributable to their infection. Risk factors for infection reflected high exposure to mosquitoes; rural residents were particularly at risk (prevalence ratio: 8.2, 95% CI: 1.1-58.7). DISCUSSION: This study adds to the evidence that WNV lineage 2 strains can cause significant illness, demonstrating ratios of infection to clinical disease similar to those found previously for WNV lineage 1.

European surveillance for West Nile virus in mosquito populations.

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.