Sentinel pigeon surveillance for West Nile virus by using lard-can traps at differing elevations and canopy cover classes

Sentinel pigeons, Columba livia, were installed in lard-can traps at heights of 1.5 m and 7.6-9.1 m within differing canopy cover classes in New York City. Adult mosquitoes were collected weekly from July to October 2002, as were serum samples from each pigeon. Culex pipiens L. and Culex restuans Theobald comprised 97% of mosquitoes collected and were most numerous in canopy-level, forested traps. The West Nile virus (family Flaviviridae, genus Flavivirus, WNV) seroconversion rate was significantly greater for pigeons in canopy-level traps, although seroconversions occurred concurrently with human cases in the city and were of little prognostic value to public health agencies. Our results indicate that sentinel pigeons were most effective for monitoring enzootic transmission of WNV when placed in single-sentinel caging 7.6-9.1 m above ground level.     

Evaluation of potential West Nile virus vectors in Volgograd region, Russia, 2003 (Diptera: Culicidae): species composition, bloodmeal host utilization, and virus infection rates of mosquitoes

Potential West Nile virus (family Flaviviridae, genus Flavivirus, WNV) vectors were assessed during 2003 at indoor and outdoor collection sites in urban Volgograd, Russia, and in three nearby towns and surrounding rural areas. In total, 9,182 female mosquitoes comprising 13 species in six genera were collected. Relative abundance and bloodmeal host utilization differed temporarily and spatially. During June and July in Volgograd, Aedes vexans (Meigen) (85.4%) and Culex p. pipiens L. (7.6%) were the two most abundant species collected indoors, whereas during August, Cx. p. pipiens was the dominant species, accounting for 87.9% of specimens collected. Two WNV-positive mosquito pools were detected in August: one pool was composed of Cx. p. pipiens and the other pool of Culex modestus Ficalbi. Anopheles messeae Falleroni, Aedes caspius (Pallas), Ae. vexans, Cx. modestus, and Cx. p. pipiens used both humans and birds as bloodmeal sources. In urban areas, 20.4% of the Cx. p. pipiens fed on humans, 58.1% fed on chickens, and six specimens were positive for both chicken and human blood. Culex p. pipiens collected from flooded basements were predominantly autogenous (91.7%), whereas adult females resting in buildings with dry basements were composed of 67.5% anautogenous and 32.5% autogenous specimens. Our data suggest that the primary WNV vectors in the Volgograd region were Cx. p. pipiens and Cx. modestus and that intense transmission of WNV to humans in urban areas during the epidemic of 1999 may have been facilitated by the abundance and concentration of anautogenous Cx. p. pipiens in multistory buildings. The role of autogenous Cx. p. pipiens in urban transmission remains unresolved.     

Effectiveness of mosquito traps in measuring species abundance and composition

Mosquito species abundance and composition estimates provided by trapping devices are commonly used to guide control efforts, but knowledge of trap biases is necessary for accurately interpreting results. We tested the hypothesis that commercially available traps (Mosquito Magnet-Pro, the Mosquito Magnet-X) would be significant improvements over the CDC Miniature Light Trap with respect to abundance, species diversity, and measures of recruitment in a wooded area of the Bronx Zoo in New York City, NY. The Mosquito Magnet-Pro collected significantly more mosquitoes (n = 1,117; mean per night, 124 +/- 28.3) than the CDC Miniature Light Trap (n = 167; mean per night, 19 +/- 5.5). The Simpson's diversity index was greatest for the Mosquito Magnet-Pro. A CDC light trap from a simultaneous surveillance project was located 15 m away and used as a control trap to test for significant differences in mosquito counts on nights with or without the experimental traps. There were no significant differences between nights, indicating the test traps did not recruit beyond 15 m. The traps differed significantly in abundance, but they had similarly limited sampling areas. Measured differences in abundance were independent of differences in diversity. This study highlights how differences between traps might affect species abundance and composition estimates.     

Ability of selected Kenyan mosquito (Diptera: Culicidae) species to transmit West Nile virus under laboratory conditions

West Nile virus (WNV) is currently active in Kenya as evidenced by the detection of antibodies in birds bled as part of an avian influenza surveillance program in 2009. Although WNV has been isolated from several mosquito species in Kenya, no studies have ever been conducted to determine which of these species are competent vectors of this virus. Therefore, we allowed Kenyan mosquitoes to feed on 2- or 3-d-old chickens that had been infected with a Lineage one strain of WNV 24-48 h earlier. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. All five species [Culex quinquefasciatus Say, Culex univittatus Theobald, Culex vansomereni Edwards, Mansonia africana (Theobald), and Mansonia uniformis (Theobald)] were susceptible to infection, but disseminated infections were detected only in the three Culex, and not the two Mansonia species. Culex mosquitoes with a disseminated infection readily transmitted virus by bite, but even when inoculated with WNV, the two Mansonia failed to transmit virus, indicating a salivary gland barrier. These studies indicate that the three Culex species may play a role in the transmission of WNV in Kenya.     

A naturally occurring mutation within the probe-binding region compromises a molecular-based West Nile virus surveillance assay for mosquito pools (Diptera: Culicidae)

A naturally occurring mutation was detected within the probe binding region targeting the envelope gene sequence of West Nile virus used in real-time polymerase chain reaction assays to test mosquito pools and other samples. A single C-->T transition 6nt from the 5' end of the 16mer in the envelope gene probe-binding region at genomic position 1,194 reduced assay sensitivity. The mutation first was detected in 2009 and persisted at a low prevalence into 2011. The mutation caused a 0.4% false negative error rate during 2011. These data emphasized the importance of confirmational testing and redundancy in surveillance systems relying on highly specific nucleic acid detection platforms.     

Host-feeding patterns of Culex pipiens and other potential mosquito vectors (Diptera: Culicidae) of West Nile virus (Flaviviridae) collected in Portugal

The host blood-feeding patterns of mosquito vectors affects the likelihood of human exposure to zoonotic pathogens, including West Nile Virus (family Flaviviridae, genus Flavivirus, WNV). In Portugal, data are unavailable regarding the blood-feeding habits of common mosquito species, including Culex pipiens L., considered the primary vector of WNV to humans. The sources of bloodmeals in 203 blood-fed mosquitoes of nine species collected from June 2007 to November 2010 in 34 Portuguese counties were analyzed by sequencing cytochrome-b partial fragments. Cx. pipiens was the most common species collected and successfully analyzed (n = 135/78). In addition, blood-fed females of the following species were analyzed: Ochlerotatus caspius Pallas (n = 20), Culex theileri Theobald (n = 16), Anopheles maculipennis s.l. Meigen (n = 10), Culiseta longiareolata Macquart (n = 7), Aedes aegypti L. (n = 6), Culex perexiguus Theobald (n = 3), Culiseta annulata Schrank (n = 3), and Ochlerotatus detritus Haliday (n = 3). The Cx. pipiens mosquitoes fed predominantly on birds (n = 55/78, 70.5%), with a high diversity of avian species used as hosts, although human blood was identified in 18 specimens (18/78, 23.1%). No significant differences were found between the host-feeding patterns of blood-fed Cx. pipiens collected in residential and nonresidential habitats. The occurrence of human derived blood meals and the presence of a mix avian-human bloodmeal accordingly suggest this species as a potential vector of WNV. Therefore, in Portugal, Cx. pipiens may play a role both in the avian-to-avian enzootic WNV cycle and in the avian-to-mammal transmission. In this context, the identity of Cx. pipiens (considering the forms molestus and pipiens) and the potential consequence on feeding behavior and WNV transmission are discussed.     

Theoretical formulation for mosquito host-feeding patterns: application to a West Nile virus focus of southern France

Host-feeding patterns play a key role in the transmission of vector-borne diseases such as West Nile fever, which involves two kinds of vertebrates, birds and mammals. In this study, we propose a theoretical formulation for mosquito host-feeding patterns using three quantities, as follows: the apparent attractiveness/contact probabilities, the conditional host(-feeding) preferences, and the enzootic versus bridge probabilities. Using results from host-baited trap collections, the quantities defined above were assessed for the most abundant mosquito species in the main West Nile virus focus of southern France. We found that host availability is important in determining the efficiency of bridge vectors, and that even ornithophilic mosquitoes like Culex species, classically classified as enzootic vectors, may turn out to be efficient bridge vectors in certain contexts of host abundance. Our developed theoretical framework can easily be adapted and applied to other experimental data and other vector-borne diseases.     

Resistance of malaria vectors to pyrethrins used for impregnating mosquito nets in Benin, West Africa]

Impregnated bednets can be considered a major tool for reducing Anopheles bites, malaria morbidity and overall mortality. The resistance of Anopheles gambiae to pyrethroids used to impregnate bednets and curtains has already been noted in the urban area of Cotonou in Benin (18, 21). In this study, we wished to find out if the resistance observed in Cotonou is localized only in this town or is already extensive throughout Benin. In this case, such resistance would be a handicap to the promotion of impregnated bednets in Benin. The study was carried out in 15 localities throughout the different ecological zones of Benin. The study has also taken into account environmental factors favouring the emergence of resistance. We did susceptibility tests with WHO test kits for adult mosquitoes using impregnated papers. The papers were impregnated with permethrin 0.25%, deltamethrin 0.025% and lambdacyhalothrin 0.1%. We also tested DDT 4% to find out if there was a cross resistance between DDT and the pyrethroids. Two mosquito species were tested: An. gambiae and An melas. In northern Benin, where farmers use insecticides against cotton pests, vectors are susceptible to deltamethrin and lambdacyhalothrin and resistant to permethrin. In the south, An. gambiae is resistant to deltamethrin and permethrin. This resistance is high in the urban zone of Cotonou, in the coastal and lagoon areas and at Kraké, a frontier viliage with Nigeria. The resistance observed in southern Benin is confirmed by the lengthening of the knock-down time of mosquitoes which were exposed for 1 hour to insecticide in impregnated WHO test tubes, and by a reduction of permethrin and deltamethrin remanence effect.     

Viral pathogenesis in mice is similar for West Nile virus derived from mosquito and mammalian cells

West Nile virus (WNV) is a mosquito-borne pathogen. During replication, WNV acquires different carbohydrates and lipid membranes, depending on its mosquito or vertebrate hosts. Consequently, WNV derived from mosquito and vertebrate cell lines differ in their infectivity for dendritic cells (DCs) and induction of type I interferon (IFN-α/β) in vitro. We evaluated the pathogenesis of WNV derived from mosquito (WNV^C6/36) and vertebrate (WNV^BHK) cell lines in mice. The tissue tropism, infectivity, clinical disease, and mortality did not differ for mice inoculated with WNV^C6/36 or WNV^BHK, and there were only minor differences in viral load and serum levels of IFN-α/β. The replication kinetics of WNV^C6/36 and WNV^BHK were equivalent in primary DCs and skin cells although primary DCs were more susceptible to WNV^C6/36 infection than to WNV^BHK infection, suggesting that less virus is produced per infected cell for WNV^C6/36. In conclusion, viral source has minimal effect on WNV pathogenesis in vivo.     

Neutralization escape variant of West Nile virus associated with altered peripheral pathogenicity and differential cytokine profile

In order to understand the factors influencing pathogenicity of a virus, two neutralization escape (NE) variants were selected from wild type lineage 1 West Nile virus (WNV) 68856 strain pathogenic by intra-peritoneal (i.p.) route using monoclonal antibodies (MAbs) against envelope (E) protein. Both NE IF1A7 1.1 and NE IVC3F10 1.2 were resistant to neutralization and were neurovirulent by intra-cranial (i.c.) inoculation. Growth kinetics in porcine stable (PS) kidney and baby hamster kidney (BHK) cells was unchanged. In contrast to parent WNV only NE IF1A7 1.1 failed to cause lethal encephalitis on i.p. inoculation and was non pathogenic. NE IF1A7 1.1 variant showed delayed replication kinetics in murine peritoneal exudate cells (PEC) and Neuro 346 cells in vitro. In comparison with parent WNV and NE IVC3F10 1.2 variant, non pathogenic variant exhibited significantly reduced tumour necrosis factor α (TNF-α) induction in infected animals and PEC. Other cytokines like Interleukin (IL)-10, IL-6 and Interferon (IFN)-β remained unchanged. However, IL-1β did not follow the pattern and was higher only in parent WNV-infected PEC. The E gene sequences of these NE variants showed three common amino acid substitutions at residues E50, E89 and E242. A unique E156 (ser→pro) substitution in NE IF1A7 1.1, was absent in NE IVC3F10 1.2 variant suggested probable virulence marker. Our data indicates possible role of WNV E protein in induction of TNF-α and IL-1β and its association with WNV pathogenesis.     

West Nile Virus: Biology,Transmission, and Human Infection

Summary: West Nile Virus was introduced into the Western Hemisphere during the late summer of 1999 and has been causing significant and sometimes severe human diseases since that time. This article briefly touches upon the biology of the virus and provides a comprehensive review regarding recent discoveries about virus transmission, virus acquisition, and human infection and disease.     

Chemical composition and larvicidal evaluation of Mentha, Salvia, and Melissa essential oils against the West Nile virus mosquito Culex pipiens

The volatile metabolites of wild-growing Mentha spicata, M. longifolia, M. suaveolens, Melissa officinalis, Salvia fruticosa, S. pomifera subsp. calycina, and S. pomifera subsp. pomifera from Greece were determined by gas chromatography and gas chromatography–mass spectrometry. The insecticidal properties of the analyzed essential oils were screened on Culex pipiens larvae. Additionally two of the main components of the essential oils, piperitenone oxide and 1,8-cineole were assayed against C. pipiens in order to define the affiliation between them and the larvicidal properties of the oils. The most effective oils were M. suaveolens (major constituent piperitenone oxide, 62.4%), M. spicata (piperitenone oxide, 35.7% and 1,8-cineole, 14.5%) and M. longifolia—Central Greece (piperitenone oxide, 33.4%; 1,8-cineole, 24.5% and trans-piperitone epoxide, 17.4%), which exhibited LC₅₀ values ranging from 47.88 to 59.33 mg l⁻¹. Medium activity revealed the oils of M. officinalis (terpin-4-ol, 15.8%; caryophyllene oxide, 13.2%; sabinene, 12.9%; β-pinene, 12.1%; and trans-caryophyllene, 10.2%), M. longifolia—Southern Greece (carvone, 54.7% and limonene 20.0%), S. pomifera subsp. pomifera (trans-caryophyllene, 22.5% and trans-thujone, 21.0%), S. pomifera subsp. calycina—West Southern Greece (trans-thujone, 56.1% and 1,8-cineole, 10.4%), and S. fruticosa—population 2 (camphor, 23.1%; α-pinene, 12.7%; and borneol, 12.6%), with LC₅₀ values ranging from 78.28 to 91.45 mg l⁻¹. S. pomifera subsp. calycina (Central Greece) essential oil (trans-thujone, 26.5% and cis-thujone, 12.0%) presented rather low activity (LC₅₀ values 140.42 mg l⁻¹), while S. fruticosa—population 1 (1,8-cineole, 31.4% and camphor, 22.6%) was the only inactive oil. Additionally, the constituent piperitenone oxide was found to be highly active (LC₅₀ values 9.95 mg l⁻¹), whereas 1,8-cineole revealed no toxicity.     

Larvicidal activity of selected plant hydrodistillate extracts against the house mosquito, Culex pipiens, a West Nile virus vector

The larvicidal activity of hydrodistillate extracts from Chrysanthemum coronarium L., Hypericum scabrum L., Pistacia terebinthus L. subsp. palaestina (Boiss.) Engler, and Vitex agnus castus L. was investigated against the West Nile vector, Culex pipiens L. (Diptera: Culicidae). Yield and identification of the major essential oils from each distillation was determined by GC-MS analyses. The major essential oil component for each plant species was as follows: α-pinene for P. terebinthus palaestina, and H. scabrum (45.3% and 42.3%, respectively), trans-β-caryophyllene for V. agnus castus (22.1%), and borneol for C. coronarium (20.9%). A series of distillate concentrations from these plants (that ranged from 1 ppm to 500 ppm, depending on plant species) were assessed against late third to early fourth C. pipiens larvae at 1, 6, and 24 h posttreatment. In general, larval mortality to water treated with a distillate increased as concentration and exposure time increased. H. scabrum and P. terebinthus palaestina were most effective against the mosquito larvae and both produced 100% mortality at 250 ppm at 24-h continuous exposure compared with the other plant species. Larval toxicity of the distillates at 24 h (LC₅₀ from most toxic to less toxic) was as follows: P. terebinthus palaestina (59.2 ppm) > H. scabrum (82.2 ppm) > V. agnus castus (83.3 ppm) > C. coronarium (311.2 ppm). But when LC₉₀ values were compared, relative toxicity ranking changed as follows: H. scabrum (185.9 ppm) > V. agnus castus (220.7 ppm) > P. terebinthus palaestina (260.7 ppm) > C. coronarium (496.3 ppm). Extracts of native Turkish plants continue to provide a wealth of potential sources for biologically active agents that may be applied against arthropod pests of man and animals.     

Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus

West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.     

Chemical composition, larvicidal evaluation, and adult repellency of endemic Greek Thymus essential oils against the mosquito vector of West Nile virus

Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory–secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank’s balanced salt solution (HBSS). Concurrently, the effect of adding l-cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P > 0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only onefold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.     

West Nile virus in the United States: guidelines for detection, prevention, and control

The epidemic/epizootic of West Nile (WN) encephalitis in the northeastern United States in the summer and fall of 1999 was an unprecedented event, underscoring the ease with which emerging infectious pathogens can be introduced into new geographic areas in today's era of rapid transportation and increased movement of people, animals, and commodities. This epidemic/epizootic and the increased frequency of other exotic pathogens being imported into the United States raises the issue of whether local, state, and national public health agencies are prepared to deal with epidemics/epizootics of vector-borne infectious diseases. The overwintering of WN virus and the epizootic transmission in the summer of 2000 reinforces the need to rebuild the public health infrastructure to deal with vector-borne diseases in this country. This article summarizes guidelines for surveillance, prevention, and control of WN virus that were drafted in December 1999 to help prepare state and local health departments for monitoring WN virus activity in the spring and summer of 2000 and also summarizes the data collected from those surveillance systems through September 2000.     

Sexual differences in larval molting rates in a protandrous mosquito (Diptera: Culicidae) species, Aedes sierrensis

Day-old larval Aedes sierrensis collected from six newly flooded treeholes in northern California were reared individually in the laboratory under simulated field conditions to compare larval developmental rates of males and females. Time to adult eclosion ranged from 133 to 219 d for this generally univoltine, winter-developing species. Males experienced significantly shorter first, second, and third larval instars than females. Females spent significantly less time as fourth instars (whose endpoint is determined by photoperiod). Length of pupal stage was equal for males and females. Time to mean adult eclosion differed among treeholes but was not determined by latitudinal position of treehole. Wing lengths were shorter for males than females in this sexually dimorphic species and also differed significantly among treeholes. Wing lengths were significantly correlated with total developmental time, but females spending more time in the fourth instar did not emerge as larger adults. In natural treeholes, resource utilization during rapid development by Ae. sierrensis males may limit the size and number of females produced from the same cohort if resources are limiting.     

Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species

Three California Culex species previously identified as efficient laboratory vectors of West Nile (WN) virus were tested for their capability to vertically transmit WN virus. Wild-caught Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, and two populations of Culex tarsalis Coquillett females were inoculated intrathoracically with 10(2.7 +/- 0.1) plaque-forming units of WN virus. F1 progeny were reared at 18 degrees C and subsequently tested as adults for infectious virus on Vero cell culture. Virus was not detected in 197 pools comprising 4,884 Cx. p. pipiens. The minimum filial infection rate (MFIR) for Cx. p. quinquefasciatus was approximately 3.0/1,000 for 665 progeny tested in 28 pools. There was no virus detected in 102 pools of 2,453 progeny from Cx. tarsalis collected in Riverside County. The MFIR for Cx. tarsalis collected in Yolo County was approximately 6.9/1,000 for 2,165 progeny tested in 86 pools. Mosquito progeny infected vertically during the fall could potentially serve as a mechanism for WN virus to overwinter and initiate horizontal transmission the following spring.