Zika Virus Replication in a Mast Cell Model is Augmented by Dengue Virus Antibody-Dependent Enhancement and Features a Selective Immune Mediator Secretory Profile.

Zika virus and dengue virus are evolutionarily related and structurally similar mosquito-borne Flaviviruses. These congruencies can lead to cross-reactive antibody binding, whereby antibodies generated from previous dengue virus immunity can augment Zika virus replication in vitro. This phenomenon, termed antibody-dependent enhancement, may participate in the clinical manifestations detected in areas with Flavivirus cocirculations where Zika virus is endemic; however, a causal relationship has yet to be determined. The KU812 mast cell/basophil line was integral in identifying the first Flavivirus infection in mast cells and serves as an effective in vitro model to study dengue virus antibody-dependent enhancement. Mast cells, sentinel white blood cells intrinsic in coordinating early immune defenses, are characteristically situated in the intradermal space and are therefore among the first immune cells interfaced with blood-feeding mosquitoes. Here, we tested whether KU812 cells were permissive to Zika virus, how previous dengue virus immunity might augment Zika virus infection, and whether either condition induces an immunological response. We report an antibody-dependent enhancement effect of Zika virus infection in KU812 cells across multiple time points (48, 72, and 96 hours postinfection [hpi]) and a range of multiplicities of infection (4.0 × 10-3 to 4) using various concentrations of cross-reactive dengue virus monoclonal antibodies (D11C and 1.6D). This antigen-specific antibody-mediated infection was selectively coupled to chemokine ligand 5 (CCL5), interleukin 1ß (IL-1ß), and C-X-C motif chemokine ligand 10 (CXCL10) secretion and a reduction in granzyme B (GrB) release. Therefore, mast cells and/or basophils may significantly augment Zika virus infection in the context of preexisting dengue virus immunity. IMPORTANCE Antibodies generated against one dengue serotype can enhance infection of another by a phenomenon called antibody-dependent enhancement (ADE). Additionally, antigenic similarities between Zika and dengue viruses can promote Zika virus infection by way of ADE in vitro using these very same anti-dengue antibodies. We used the KU812 cell line to demonstrate for the first time that anti-dengue antibodies enhanced infectious Zika virus replication in a mast cell model and specifically increased CCL5, CXCL10, and IL-1ß, while also impairing granzyme B secretion. Furthermore, enhanced Zika virus infection and selective mediator release were mechanistically dependent on fragment crystallizable gamma receptor II (FcγRII). These findings establish a new model for Zika virus research and a new subcategory of immune cells previously unexplored in the context of Zika virus enhancement while being some of the very first immune cells likely to meet a blood-feeding infected mosquito.

Geospatial Analysis and Seasonal Distribution of West Nile Virus Vectors (Diptera: Culicidae) in Southern Ontario, Canada.

The purpose of this study was to establish geospatial and seasonal distributions of West Nile virus vectors in southern Ontario, Canada using historical surveillance data from 2002 to 2014. We set out to produce mosquito abundance prediction surfaces for each of Ontario's thirteen West Nile virus vectors. We also set out to determine whether elevation and proximity to conservation areas and provincial parks, wetlands, and population centres could be used to improve our model. Our results indicated that the data sets for Anopheles quadrimaculatus, Anopheles punctipennis, Anopheles walkeri, Culex salinarius, Culex tarsalis, Ochlerotatus stimulans, and Ochlerotatus triseriatus were not suitable for geospatial modelling because they are randomly distributed throughout Ontario. Spatial prediction surfaces were created for Aedes japonicus and proximity to wetlands, Aedes vexans and proximity to population centres, Culex pipiens/restuans and proximity to population centres, Ochlerotatus canadensis and elevation, and Ochlerotatus trivittatus and proximity to population centres using kriging. Seasonal distributions are presented for all thirteen species. We have identified both when and where vector species are most abundant in southern Ontario. These data have the potential to contribute to a more efficient and focused larvicide program and West Nile virus awareness campaigns.

West Nile virus in Ontario, Canada: A twelve-year analysis of human case prevalence, mosquito surveillance, and climate data.

West Nile Virus (WNV) first arrived in Ontario, Canada in 2001 and has since spread throughout most of the province, causing disease in humans. The provincial government established a province-wide surveillance program to monitor WNV transmission throughout the 36 regional health units. Here we have acquired records of WNV human and mosquito surveillance from 2002 to 2013 to describe seasonal and geographic trends in WNV activity in southern Ontario. Additionally, we obtained climate data from seven municipalities to investigate how temperature and precipitation affect WNV transmission dynamics. We identified a strong quadratic relationship between the number of confirmed human cases and positive Culex mosquito pools recorded at the end of each year (R2 = 0.9783, p < 0.001). Using Spearman rank correlation tests, we identified that the minimum infection rate of Culex pipiens/restuans pools are the strongest predictor of human cases at a 1 week lag period. We also identified positive correlations between minimum infection rates, temperature, vector abundance, and cumulative precipitation. Global Moran's I index indicates strong positive autocorrelation and clustering of positive Culex pool counts in southern Ontario. Local indicators of spatial association tests revealed a total of 44 high-high and 1 high-low trap locations (n = 680). In the current work we have identified when and where hot spots of WNV activity have occurred in southern Ontario. The municipalities surrounding the western shore of the Lake Ontario and Windsor-Essex County have the largest records of positive mosquitoes and human cases. We identified that positive mosquitoes are a strong indicator of human cases to follow in the coming weeks. An epidemic action threshold of cumulative positive Culex pools was established, allowing Ontario public health officials to predict an epidemic at epidemiological week 34 (rho = 0.90, p < 0.001). These data have the potential to contribute to more efficient larvicide programs and awareness campaigns for the public.

Zika Virus: Quantification, Propagation, Detection, and Storage.

Zika virus (ZIKV), belonging to the family Flaviviridae, genus Flavivirus, is an arthropod-borne virus that was first discovered from the Zika forest in Uganda in 1947. Recent outbreaks in South America have linked ZIKV to cases of microcephaly and Guillain-Barré syndrome in humans. With the increased interest in ZIKV, protocols must be established to facilitate proper research. Here we describe the laboratory techniques required to quantify, propagate, and store ZIVK. We also review the proper safety protocol for the handling of ZIKV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2016 by John Wiley & Sons, Inc.

A checklist of the 67 mosquito species of Ontario, Canada.

We provide an updated checklist of 67 endemic mosquito species known from Ontario, Canada. Nine endemic species are added to the checklist found in Darsie and Ward (2005) : Aedes cantator, Ae. churchillensis, Ae. nigripes, Ae. pullatus, Anopheles perplexens, An. crucians, An. smaragdinus, Culex erraticus, and Cx. salinarius. Only 4 specimens of Ae. albopictus have been recorded in Ontario since 2001 despite concerted efforts to find this species; therefore, it is considered an "accidental" species and is excluded from the checklist.

Characterization of larval habitats of Anopheles albimanus, Anopheles pseudopunctipennis, Anopheles punctimacula, and Anopheles oswaldoi s.l. populations in lowland and highland Ecuador.

Recent collection data indicate that at least four potential malaria vectors occupy more widespread distributions within the Andean highlands than in the past. Since habitat elimination is an important aspect of malaria control, it is vital to characterize larval habitats for Anopheles species within both lowland and highland sites. To that end, 276 sites within Ecuador were surveyed between 2008 and 2010. Characteristics of Anopheles-present sites for four species were compared to Anopheles-absent sites within the same geographical range and also to Anopheles-absent sites within a highland range representing potential future habitats. Thermochron iButtons(©) were used to describe the daily temperature variation within a subset of potential habitats. Anopheles albimanus (W.) was positively associated with permanent habitats, sand substrates, floating algae (cyanobacterial mats), and warmer temperatures in both comparisons. Anopheles pseudopunctipennis (T.) was associated with floating algae (cyanobacterial mats), warmer temperatures, and higher water clarity in both comparisons. Anopheles punctimacula (D.&K.) was negatively associated with floating algae and positively associated with dissolved oxygen in both comparisons. Anopheles oswaldoi s.l. (P.) was not significantly associated with any parameters more often than expected given larval-absent sites. The results indicate that minimum water temperatures might limit the upper altitudinal distribution of An. albimanus (18.7° C) and An. pseudopunctipennis (16.0° C).

Malaria in highlands of Ecuador since 1900.

A recent epidemic of malaria in the highlands of Bolivia and establishment of multiple Anopheles species mosquitoes in the highlands of Ecuador highlights the reemergence of malaria in the Andes Mountains in South America. Because malaria was endemic to many highland valleys at the beginning of the 20th century, this review outlines the 20th century history of malaria in the highlands of Ecuador, and focuses on its incidence (e.g., geographic distribution) and elimination from the northern highland valleys of Pichincha and Imbabura and the role of the Guayaquil to Quito railway in creating highland larval habitat and inadvertently promoting transportation of the vector and parasite. Involvement of control organizations in combating malaria in Ecuador is also outlined in a historical context.

Larval Habitat Associations with Human Land Uses, Roads, Rivers, and Land Cover for Anopheles albimanus, A. pseudopunctipennis, and A. punctimacula (Diptera: Culicidae) in Coastal and Highland Ecuador.

Larval habitat for three highland Anopheles species: Anopheles albimanus Wiedemann, Anopheles pseudopunctipennis Theobald, and Anopheles punctimacula Dyar and Knab was related to human land uses, rivers, roads, and remotely sensed land cover classifications in the western Ecuadorian Andes. Of the five commonly observed human land uses, cattle pasture (n = 30) provided potentially suitable habitat for A. punctimacula and A. albimanus in less than 14% of sites, and was related in a principal components analysis (PCA) to the presence of macrophyte vegetation, greater surface area, clarity, and algae cover. Empty lots (n = 30) were related in the PCA to incident sunlight and provided potential habitat for A. pseudopunctipennis and A. albimanus in less than 14% of sites. The other land uses surveyed (banana, sugarcane, and mixed tree plantations; n = 28, 21, 25, respectively) provided very little standing water that could potentially be used for larval habitat. River edges and eddies (n = 41) were associated with greater clarity, depth, temperature, and algae cover, which provide potentially suitable habitat for A. albimanus in 58% of sites and A. pseudopunctipennis in 29% of sites. Road-associated water bodies (n = 38) provided potential habitat for A. punctimacula in 44% of sites and A. albimanus in 26% of sites surveyed. Species collection localities were compared to land cover classifications using Geographic Information Systems software. All three mosquito species were associated more often with the category "closed/open broadleaved evergreen and/or semi-deciduous forests" than expected (P ≤ 0.01 in all cases), given such a habitat's abundance. This study provides evidence that specific human land uses create habitat for potential malaria vectors in highland regions of the Andes.

New highland distribution records of multiple Anopheles species in the Ecuadorian Andes.

BACKGROUND: Several recent climate change reviews have stressed the possibility of some malaria vectors occupying regions of higher altitudes than previously recorded. Indeed, highland malaria has been observed in several African nations, possibly attributable to changes in land use, vector control and local climate. This study attempts to expand the current knowledge of the distribution of common Anopheles species in Ecuador, with particular attention to highland regions (> 500 m) of the Andes. METHODS: Extensive field collections of larvae were undertaken in 2008, 2009 and 2010 throughout all regions of Ecuador (except the lower-altitude Amazonian plain) and compared to historical distribution maps reproduced from the 1940s. Larvae were identified using both a morphological key and sequencing of the 800 bp region of the CO1 mitochondrial gene. In addition, spatial statistics (Getis-Ord Hotspot Analysis: Gi*) were used to determine high and low-density clusters of each species in Ecuador. RESULTS: Distributions have been updated for five species of Anopheles in Ecuador: Anopheles albimanus, Anopheles pseudopunctipennis, Anopheles punctimacula, Anopheles eiseni and Anopheles oswaldoi s.l.. Historical maps indicate that An. pseudopunctipennis used to be widespread in highland Andean valleys, while other species were completely restricted to lowland areas. By comparison, updated maps for the other four collected species show higher maximum elevations and/or more widespread distributions in highland regions than previously recorded. Gi* analysis determined some highland hot spots for An. albimanus, but only cold spots for all other species. CONCLUSIONS: This study documents the establishment of multiple anopheline species in high altitude regions of Ecuador, often in areas where malaria eradication programs are not focused.

Malaria Knowledge, Concern, Land Management, and Protection Practices among Land Owners and/or Managers in Lowland versus Highland Ecuador.

To control malaria effectively, it is essential to understand the current knowledge, beliefs, concerns, land management practices, and mosquito bite protection methods in use by citizens. This study presents a comparative, quantitative, interview-based study of land owners and/or managers (n = 262) in the Ecuadorian lowlands (presently considered malarious) (n = 131) and highlands (potentially malarious in the future) (n = 131). Although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water, mosquitoes, and malaria, about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property. As well, about half of respondents with potential anopheline larval habitat did not report its presence, likely due to a highly specific definition of suitable mosquito habitat. Most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention, most commonly bed nets.

A modified Centers for Disease Control and Prevention gravid trap for easier mosquito collection.

The original Centers for Disease Control and Prevention (CDC) gravid trap, with its mesh collection bag, can cause considerable damage to the captured mosquitoes. In an effort to reduce this damage and keep costs at a minimum, we modified our existing CDC gravid traps to accept a collection cup from a CDC light trap. This modification allowed for easier transport, freezing, storage, and removal of the mosquitoes. Modifying the CDC gravid traps takes little expertise or resources and can be completed quickly.

Establishment of Ochlerotatus japonicus (Diptera: Culicidae) in Ontario, Canada.

Ochlerotatus (Finlaya) japonicus (Theobold) is newly established in Ontario, Canada. It was first discovered in 2001 during the province-wide West Nile virus mosquito surveillance program implemented by the Ontario Ministry of Health and Long Term Care. Although the numbers of adults trapped in CDC light traps were low in 2001 and 2002, they increased during the 2003 and 2004 surveillance seasons. Oc. japonicus larvae also have been collected in large numbers in the Niagara Peninsula in a variety of natural and artificial containers. The number of health units with records for Oc. japonicus has increased over the 4 yr of surveillance, illustrating the ability of this species to rapidly extend its range. As a potential arboviral bridge vector, its establishment in Ontario requires further study and should be considered a public health concern.

Attraction of Culex pipiens/restuans (Diptera: Culicidae) mosquitoes to bird uropygial gland odors at two elevations in the Niagara region of Ontario.

In an effort to determine whether female Culex pipiens L. and Culex restuans Theobald mosquitoes (Diptera: Culicidae) are attracted to crow, Corvus brachyrhynchus, uropygial gland secretions, CDC miniature light traps (baited with CO2 but with the lights removed) were placed at approximately 1.5- and 5-m elevations, in 10 trees in awoodlot near Niagara Falls, Canada. These traps were assigned either a bird odor or a blank control. Bird odors were created by attaching cotton swabs coated with crow uropygial gland secretions to the trap intake. A significantly greater number of Cx. pipiens/ restuans were found in the 5-m traps compared with 1.5-m traps, with a significant number attracted to the bird odor over the no odor traps at the 5-m elevation, but not at 1.5 m. We also found more Aedes vexans (Meigen) in the 1.5-m traps than the 5-m traps; however, presence or absence of bird odor did not influence the distribution of Ae. vexans.

Polytene chromosomes of an archipelagic subgenus, Inseliellum (Diptera: Simuliidae).

The black fly subgenus Inseliellum is present on a series of archipelagos in the South Pacific. In this study, larval polytene chromosome maps of six Inseliellum species are presented. Chromosomal relationships among taxa were determined through shared fixed inversions or chromosomal landmark positioning. Three fixed inversions (IL-2, IIS-1, and IIIS-1) were shared among species, as was the position of the nucleolar organizer (NO) (IL or IIL). Comparisons to two previously studied species of Inseliellum are included to produce a cytological transformation series among eight taxa. The NO position defines two clades in the phylogeny of Inseliellum, herein named the NO-IL and NO-IIL clades. The utility of this cytological data set is discussed.

The physical gene Hsp70 map on polytene chromosomes of Anopheles darlingi from the Brazilian Amazon.

In situ hybridization was used to determine the physical location of the Hsp70 genes in salivary polytene chromosomes of Anopheles darlingi from Manaus and Macapá, Brazil, and to assess the usefulness of the Hsp70 locus as a genetic marker in A. darlingi populations. In both populations, the double markings corresponding to the Hsp70-12A and Hsp70-14A genes were located on the right arm of chromosome 2. The Hsp70 locus was considered to be an excellent marker for studying chromosomal evolution and relationships among A. darlingi populations.

West Nile virus infection rates in pooled and individual mosquito samples.

The detection of West Nile virus (WNV) in mosquitoes by real-time RT-PCR provides valuable information on the epidemiology of the virus and identifies mosquito species that are potential vectors. Testing sets of pooled mosquitoes of the same species is logistically the easiest and most cost-effective approach for WNV testing; however, little information is available on how the results of small pooled sets relate to those of testing individual mosquitoes. During the 2002 outbreak, we compared pooled and individual samples of two mosquito species (Culex pipiens and Culex restuans) collected from three Health Unit regions in Ontario, Canada. Significantly more Cx. restuans were infected with WNV compared to Cx. pipiens. We show that with pool sizes of five individuals both MIR (minimum infection rates) and MLE (maximum likelihood estimation) values were acceptable in estimating infection rates.

Polytene chromosomes of Simulium craigi (Diptera: Simuliidae).

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype 'CC' from Pennsylvania; cytotype 'AF' from Ontario and Manitoba; and cytotype 'ACF/BCF' from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.