Seroconversion for West Nile and St. Louis encephalitis viruses among sentinel horses in Colombia

We prospectively sampled flavivirus-na?ve horses in northern Colombia to detect West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) seroconversion events, which would indicate the current circulation of these viruses. Overall, 331 (34.1%) of the 971 horses screened were positive for past infection with flaviviruses upon initial sampling in July 2006. During the 12-month study from July 2006-June 2007, 33 WNV seroconversions and 14 SLEV seroconversions were detected, most of which occurred in the department of Bolivar. The seroconversion rates of horses in Bolivar for the period of March-June 2007 reached 12.4% for WNV and 6.7% for SLEV. These results comprise the first serologic evidence of SLEV circulation in Colombia. None of the horses sampled developed symptoms of encephalitis within three years of initial sampling. Using seroconversions in sentinel horses, we demonstrated an active circulation of WNV and SLEV in northern Colombia, particularly in the department of Bolivar. The absence of WNV-attributed equine or human disease in Colombia and elsewhere in the Caribbean Basin remains a topic of debate and speculation.     

Evidence of vertical transmission of West Nile virus in field‐collected mosquitoes

Male and nulliparous female mosquitoes were surveyed for evidence of vertical WNV infection in East Baton Rouge Parish, Louisiana. Adult male mosquitoes collected by trapping and aspiration, and adult male and nulliparous female mosquitoes reared from field‐collected larvae were tested. Adult male Culex spp., female Aedes albopictus (Skuse), and female Culex quinquifasciatus Say mosquitoes that were collected as larvae were test‐positive for WNV RNA. Infectious WNV was detected using virus isolation in field‐collected male Aedes triseriatus Say and Culex salinarius Coquillett; these data represent the first field evidence of vertical transmission of WNV in Ae. triseriatus and Cx. salinarius.     

Serological,Molecular and Entomological Surveillance Demonstrates Widespread Circulation of West Nile Virus in Turkey

West Nile virus (WNV), a mosquito-borne flavivirus with significant impact on human and animal health, has recently demonstrated an expanded zone of activity globally. The aim of this study is to investigate the frequency and distribution of WNV infections in potential vectors and several mammal and avian species in Turkey, where previous data indicate viral circulation. The study was conducted in 15 provinces across Turkey during 2011–2013. In addition, the entomological study was extended to 4 districts of the Turkish Republic of Northern Cyprus. WNV exposure was determined in humans, horses, sheep and ducks from Mersin, Sanliurfa, Van and Kars provinces of Turkey, via the detection of neutralizing antibodies. WNV RNA was sought in human and equine samples from Mersin, Adana and Mugla provinces. Field-collected mosquitoes from 92 sites at 46 locations were characterized morphologically and evaluated for viral RNA. Neutralizing antibodies were identified in 10.5% of the 1180 samples studied and detected in all species evaluated. Viral nucleic acids were observed in 5.9% of 522 samples but only in horses. A total of 2642 mosquito specimens belonging to 15 species were captured, where Ochlerotatus caspius (52.4%), Culex pipiens sensu lato (24.2%) comprise the most frequent species. WNV RNA was detected in 4 mosquito pools (1.9%), that comprise Oc. caspius Cx. pipiens s.l. and DNA barcoding revealed the presence of Cx. quinquefasciatus and Cx. perexiguus mosquitoes in infected Culex pools. All WNV partial sequences were characterized as lineage 1 clade 1a. These findings indicate a widespread WNV activity in Turkey, in Eastern Thrace and Mediterranean-Aegean regions as well as Southeastern and Northeastern Anatolia.     

Nonconsensus West Nile virus genomes arising during mosquito infection suppress pathogenesis and modulate virus fitness in vivo

West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV.     

West Nile virus experimental evolution in vivo and the trade-off hypothesis

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong.     

trans-Packaged West Nile virus-like particles: infectious properties in vitro and in infected mosquito vectors

A trans-packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 10(9) infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.     

West Nile virus cluster analysis and vertical transmission in Culex pipiens complex mosquitoes in Sacramento and Yolo Counties,California, 2011

West Nile virus (WNV) is now endemic in California, with annual transmission documented by the statewide surveillance system. Although much is known about the horizontal avian‐mosquito transmission cycle, less is known about vertical transmission under field conditions, which may supplement virus amplification during summer and provide a mechanism to infect overwintering female mosquitoes during fall. The current study identified clusters of WNV‐infected mosquitoes in Sacramento and Yolo Counties, CA, during late summer 2011 and tested field‐captured ovipositing female mosquitoes and their progeny for WNV RNA to estimate the frequency of vertical transmission. Space‐time clustering of WNV‐positive Culex pipiens complex pools was detected in the northern Elk Grove area of Sacramento County between July 18 and September 18, 2011 (5.22 km radius; p<0.001 and RR=7.80). Vertical transmission by WNV‐infected females to egg rafts was 50% and to larvae was 40%. The estimated minimal filial infection rate from WNV‐positive, ovipositing females was 2.0 infected females/1,000. The potential contribution of vertical transmission to WNV maintenance and amplification are discussed.     

Mosquito‐borne West Nile virus (WNV) surveillance in the Upper Rhine Valley,Germany

West Nile virus (WNV) could be introduced into Germany via migratory birds originating from Africa or southern Europe and subsequently transmitted to indigenous birds, humans, or horses by mosquitoes. Neither the virus itself nor antibodies against WNV have yet to be found in mosquitoes and horses, whereas antibodies have been detected in migrating birds and in humans that were in close contact with birds. At present, the West Nile virus itself has yet to be detected in Germany. This investigation was conducted primarily in major bird breeding, resting, and roosting habitats (hotspots) in the Upper Rhine Valley. Adult mosquitoes were trapped using CO₂‐baited Encephalitis Vector Surveillance (EVS)‐traps and were tested for WNV by the VecTest WNV Antigen Assay. In 2007 and 2008, a total of 11,073 host‐seeking adult female mosquitoes (13 species) were tested, and all tests were negative for WNV. Statistical calculations could be performed only where sufficient numbers of mosquitoes were trapped. For these sites, WNV infection among mosquitoes could be ruled out with 80% certainty. For the evaluation of the WNV situation in Germany, the results of this investigation are a further indication that the virus has not yet arrived.     

West Nile virus genetic diversity is maintained during transmission by Culex pipiens quinquefasciatus mosquitoes

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.     

Innate immune control of West Nile virus infection

West Nile virus (WNV), from the Flaviviridae family, is a re-emerging zoonotic pathogen of medical importance. In humans, WNV infection may cause life-threatening meningoencephalitis or long-term neurologic sequelae. WNV is transmitted by Culex spp. mosquitoes and both the arthropod vector and the mammalian host are equipped with antiviral innate immune mechanisms sharing a common phylogeny. As far as the current evidence is able to demonstrate, mosquitoes primarily rely on RNA interference, Toll, Imd and JAK-STAT signalling pathways for limiting viral infection, while mammals are provided with these and other more complex antiviral mechanisms involving antiviral effectors, inflammatory mediators, and cellular responses triggered by highly specialized pathogen detection mechanisms that often resemble their invertebrate ancestry. This mini-review summarizes our current understanding of how the innate immune systems of the vector and the mammalian host react to WNV infection and shape its pathogenesis.     

Evaluation of various attributes of gravid female traps for collection of Culex in Florida.

Gravid female traps are used for surveillance of populations of Culex spp. and arboviruses such as West Nile virus. Field trials evaluating various attributes of gravid female traps for collection of gravid Culex were conducted in rural habitats in Alachua and Marion Counties, FL. Screened cage tests in the field were also used to compare trap models. Three trap models were evaluated: two based on the original Reiter design (Gravid CDC traps A and B) and one based on the revised Reiter design (gravid box trap). Trap model significantly affected collection of Culex quinquefasciatus and Culex nigripalpus; the greatest numbers were collected by the CDC gravid trap Aand CDC gravid trap B traps compared to the gravid box traps. There were no statistically significant differences between the collections with CDC gravid traps A and B. These patterns were similar at both sites and between early and late summer collections in Marion Co. In trials conducted in screened field cages with gravid Cx. quinquefasciatus, significantly more mosquitoes were collected in the CDC gravid trap A and B than in box gravid traps. Size of pan associated with the gravid traps was important and traps with larger pans collected more mosquitoes. Pan color was also important in efficacy of traps, and traps with darker pans (black, green) collected more mosquitoes than traps with lighter pans (tan, white). The percentages of gravid mosquitoes in trap collections of Culex females ranged from 54.0% to 89.6% with no clear differences between model of trap, size of pan, or pan color. Various attributes of gravid female traps impact the efficacy of the traps for Culex spp. and these should be considered in trapping programs.     

Potential vectors of West Nile virus following an equine disease outbreak in Italy

In the late summer of 1998, an outbreak of equine encephalomyelitis due to West Nile virus (WNV) occurred in the Tuscany region of central Italy. The disease was detected in 14 race horses from nine localities in four Provinces: Firenze, Lucca, Pisa and Pistoia. The outbreak area included Fucecchio wetlands (1800 ha), the largest inland marsh in Italy, and the adjacent hilly Cerbaie woodlands with farms breeding horses. To detect potential vectors of WNV, entomological surveys of Fucecchio and Cerbaie were undertaken during 1999-2002 by collecting mosquito larvae from breeding sites and adult mosquitoes by several methods of sampling. Among 6023 mosquitoes (Diptera: Culicidae) collected, 11 species were identified: Aedes albopictus (Skuse), Ae. vexans (Meigen), Anopheles atroparvus Van Thiel, An. maculipennis Meigen s.s., An. plumbeus Stephens, Culex impudicus Ficalbi, Cx. pipiens L., Culiseta longiareolata Macquart), Ochlerotatus caspius (Pallas), Oc. detritus (Haliday) and Oc. geniculatus (Olivier). In Fucecchio marshes, Cx. impudicus predominated with seasonal peak densities in spring and autumn: its greatest abundance during early spring coincides with arrival of migratory birds from Africa. In Cerbaie hills, Cx. pipiens predominated with peak population density in late summer. No viruses were isolated from 665 mosquitoes processed. These findings, plus other data on Italian mosquito bionomics, suggest a possible mode of WNV transmission involving the most abundant Culex in the Fucecchio-Cerbaie areas. Culex impudicus, being partly ornithophilic, might transmit WNV from migratory to non-migratory birds during springtime; Cx. pipiens, having a broader host range, would be more likely to transmit WNV from birds to horses and, perhaps, to humans by late summer.     

Blood meal analysis, flavivirus screening, and influence of meteorological variables on the dynamics of potential mosquito vectors of West Nile virus in northern Italy

An extended area of northern Italy has experienced several West Nile virus (WNV) outbreaks and the emergence of Usutu virus (USUV) during previous years. Our aim was to study some of the factors that could explain disease patterns in the Trentino region, where circulation was detected in human sera and sentinel chickens, but no human or equine cases were reported. We collected Culex species (Diptera: Culicidae) in peridomestic environments. The collected specimens were analyzed for feeding behavior, the influence of temperature and rainfall on the abundance of mosquitoes, and the occurrence of flaviviruses. Analysis of blood meals showed that Culex pipiens fed mainly on blackbirds (Turdus merula) and house sparrows (Passer domesticus), while Culex hortensis fed strictly on lizards. The abundance of Cx. pipiens females correlated positively with mean temperature and negatively with rainfall (one to four weeks before capture). This negative relationship could be due to the direct effect of the flushing of habitats together with an indirect effect of oviposition repellency. The mean weekly temperature influenced the abundance of Cx. hortensis. No flaviviruses were detected in the analyzed Culex mosquitoes. These data suggest a silent cycle at low enzootic transmission levels in the area. Furthermore, we present the first contribution to understanding the transmission role of Cx. pipiens mosquitoes in Italy by identifying vertebrate hosts to species level.     

West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance

Background

West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic.

Methodology/Principal Findings

We assessed WNV vector competence of Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase.

Conclusions/Significance

Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe.     

Potential transmission of West Nile virus in the British Isles: an ecological review of candidate mosquito bridge vectors

West Nile virus (WNV) transmitted by mosquitoes (Diptera: Culicidae) infects various vertebrates, being pathogenic for birds, horses and humans. After its discovery in tropical Africa, sporadic outbreaks of WNV occurred during recent decades in Eurasia, but not the British Isles. WNV reached New York in 1999 and spread to California by 2003, causing widespread outbreaks of West Nile encephalitis across North America, transmitted by many species of mosquitoes, mainly Culex spp. The periodic reappearance of WNV in parts of continental Europe (from southern France to Romania) gives rise to concern over the possibility of WNV invading the British Isles. The British Isles have about 30 endemic mosquito species, several with seasonal abundance and other eco-behavioural characteristics predisposing them to serve as potential WNV bridge vectors from birds to humans. These include: the predominantly ornithophilic Culex pipiens L. and its anthropophilic biotype molestus Forskal; tree-hole adapted Anopheles plumbeus Stephens; saltmarsh-adapted Ochlerotatus caspius Pallas, Oc. detritus Haliday and Oc. dorsalis (Meigen); Coquillettidia richiardii Ficalbi, Culiseta annulata Schrank and Cs. morsitans (Theobald) from vegetated freshwater pools; Aedes cinereus Meigen, Oc. cantans Meigen and Oc. punctor Kirby from seasonal woodland pools. Those underlined have been found carrying WNV in other countries (12 species), including the rarer British species Aedes vexans (Meigen), Culex europaeus Ramos et al., Cx. modestus Ficalbi and Oc. sticticus (Meigen) as well as the Anopheles maculipennis Meigen complex (mainly An. atroparvus van Thiel and An. messeae Falleroni in Britain). Those implicated as key vectors of WNV in Europe are printed bold (four species). So far there is no proof of any arbovirus transmission by mosquitoes in the British Isles, although antibodies to Sindbis, Tahyna, Usutu and West Nile viruses have been detected in British birds. Neighbouring European countries have enzootic WNV and human infections transmitted by mosquito species that are present in the British Isles. However, except for localized urban infestations of Cx. pipiens biotype molestus that can be readily eliminated, there appear to be few situations in the British Isles where humans and livestock are exposed to sustained risks of exposure to potential WNV vectors. Monitoring of mosquitoes and arbovirus surveillance are required to guard the British Isles against WNV outbreaks and introduction of more anthropophilic mosquitoes such as Stegomyia albopicta (Skuse) and Ochlerotatus japonicus (Theobald) that have recently invaded Europe, since they transmit arboviruses elsewhere.     

Simultaneous circulation of two West Nile virus lineage 2 clades and Bagaza virus in the Zambezi region,Namibia

Flaviviruses include a great diversity of mosquito-borne arboviruses with epidemic potential and high global disease burden. Several flaviviruses are circulating in southern Africa affecting humans and livestock, among them West Nile virus (WNV) and Wesselsbron virus. Despite their high relevance, no arbovirus surveillance study has been conducted for more than 35 years in Namibia. In this study we assessed the diversity of flaviviruses circulating in mosquitoes in the densely populated, semi-tropical Zambezi region of north-eastern Namibia. In total, 10,206 mosquitoes were sampled in Bwabwata and Mudumu national parks and Mashi and Wuparo conservancies and screened for flavivirus infections. A high infection rate with insect-specific flaviviruses was found with 241 strains of two previously known and seven putative novel insect-specific flaviviruses. In addition, we identified ten strains of WNV in the main vector Cx. univittatus sampled in the Mashi conservancy. Surprisingly, the strains fell into two different clades of lineage 2, 2b and 2d. Further, three strains of Bagaza Virus (BAGV) were found in Cx. univittatus mosquitoes originating from Mudumu national park. Assessment of BAGV growth in different cell lines showed high replication rates in mosquito and duck cells and about 100,000fold lower replication in human, primate and rodent cells. We demonstrate a wide genetic diversity of flaviviruses is circulating in mosquitoes in the Zambezi region. Importantly, WNV and BAGV can cause outbreaks including severe disease and mortality in humans and birds, respectively. Future studies should focus on WNV and BAGV geographic distribution, as well as on their potential health impacts in and the associated social and economic implications for southern Africa.     

Fine-scale variation in vector host use and force of infection drive localized patterns of West Nile virus transmission

The influence of host diversity on multi-host pathogen transmission and persistence can be confounded by the large number of species and biological interactions that can characterize many transmission systems. For vector-borne pathogens, the composition of host communities has been hypothesized to affect transmission; however, the specific characteristics of host communities that affect transmission remain largely unknown. We tested the hypothesis that vector host use and force of infection (i.e., the summed number of infectious mosquitoes resulting from feeding upon each vertebrate host within a community of hosts), and not simply host diversity or richness, determine local infection rates of West Nile virus (WNV) in mosquito vectors. In suburban Chicago, Illinois, USA, we estimated community force of infection for West Nile virus using data on Culex pipiens mosquito host selection and WNV vertebrate reservoir competence for each host species in multiple residential and semi-natural study sites. We found host community force of infection interacted with avian diversity to influence WNV infection in Culex mosquitoes across the study area. Two avian species, the American robin (Turdus migratorius) and the house sparrow (Passer domesticus), produced 95.8% of the infectious Cx. pipiens mosquitoes and showed a significant positive association with WNV infection in Culex spp. mosquitoes. Therefore, indices of community structure, such as species diversity or richness, may not be reliable indicators of transmission risk at fine spatial scales in vector-borne disease systems. Rather, robust assessment of local transmission risk should incorporate heterogeneity in vector host feeding and variation in vertebrate reservoir competence at the spatial scale of vector-host interaction.     

Complete sequences of mitochondria genomes of Aedes aegypti and Culex quinquefasciatus and comparative analysis of mitochondrial DNA fragments inserted in the nuclear genomes

We present complete sequences of the mitochondrial genomes for two important mosquitoes, Aedes aegypti and Culex quinquefasciatus, that are major vectors of dengue virus and lymphatic filariasis, respectively. The A. aegypti mitochondrial genome is 16,655 bp in length and that of C. quinquefasciatus is 15,587 bp, yet both contain 13 protein coding genes, 22 transfer RNA (tRNA) genes, one 12S ribosomal RNA (rRNA) gene, one 16S rRNA gene and a control region (CR) in the same order. The difference in the genome size is due to the difference in the length of the control region. We also analyzed insertions of nuclear copies of mtDNA-like sequences (NUMTs) in a comparative manner between the two mosquitoes. The NUMT sequences occupy ~0.008% of the A. aegypti genome and ~0.001% of the C. quinquefasciatus genome. Several NUMTs were found localized in the introns of predicted protein coding genes in both genomes (32 genes in A. aegypti but only four in C. quinquefasciatus). None of these NUMT-containing genes had an ortholog between the two species or had paralogous copies within a genome that was also NUMT-containing. It was further observed that the NUMT-containing genes were relatively longer but had lower GC content compared to the NUMT-less paralogous copies. Moreover, stretches of homologies are present among the genic and non-genic NUMTs that may play important roles in genomic rearrangement of NUMTs in these genomes. Our study provides new insights on understanding the roles of nuclear mtDNA sequences in genome complexities of these mosquitoes.     

West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.