Flanders hapavirus in western North America.

Flanders virus (FLAV; family Rhabdoviridae) is a mosquito-borne hapavirus with no known pathology that is frequently isolated during arbovirus surveillance programs. Here, we document the presence of FLAV in Culex tarsalis mosquitoes and a Canada goose (Branta canadensis) collected in western North America, outside of the currently recognized range of FLAV. Until now, FLAV-like viruses detected in the western United States were assumed to be Hart Park virus (HPV, family Rhabdoviridae), a closely related congener. A re-examination of archived viral isolates revealed that FLAV was circulating in California as early as 1963. FLAV also was isolated in Nebraska, Colorado, South Dakota, North Dakota, and Saskatchewan, Canada. Phylogenetic analysis of the U1 pseudogene for 117 taxa and eight nuclear genes for 15 taxa demonstrated no distinct clustering between western FLAV isolates. Assuming the range of FLAV has been expanding west, these results indicate that FLAV likely spread west following multiple invasion events. However, it remains to be determined if the detection of FLAV in western North America is due to expansion or is a result of enhanced arbovirus surveillance or diagnostic techniques. Currently, the impact of FLAV infection remains unknown.

Avian malaria co-infections confound infectivity and vector competence assays of Plasmodium homopolare.

Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concomitantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare. Song sparrow (Melospiza melodia) blood infected with a low P. homopolare parasitemia was inoculated into a naïve domestic canary (Serinus canaria forma domestica). Within 5 to 10 days post infection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P. homopolare, both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P. homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P. homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P. homopolare DNA. This pattern of mixed Pcat6 and P. homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P. cathemerium lineage (Pcat8); none developed PCR detectable P. homopolare infections. These observations suggest that the original P. homopolare-infected songbird also had two un-detectable P. cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts.

LOW PREVALENCE OF WEST NILE VIRUS ANTIBODIES IN SELECT NORTHERN CALIFORNIA OWL SPECIES (2007-2014).

The objective of this study was to determine evidence of previous West Nile virus (WNV) infection in northern California owls. Owl serum samples were collected from birds presenting to a veterinary medical teaching hospital between 2007 and 2014 and were screened for the presence of WNV antibodies by an indirect enzyme immunoassay (EIA). Only one of 71 samples (1.41%) tested was positive by EIA and confirmed by a plaque reduction neutralization test; it was the most recent sample collected. The reason for the low prevalence of WNV in these California owls despite a high prevalence in sympatric avian species in the same region is unknown and should be a topic for further research.

Reemergence of St. Louis Encephalitis Virus, California, 2015.

St. Louis encephalitis virus infection was detected in summer 2015 in southern California after an 11-year absence, concomitant with an Arizona outbreak. Sequence comparisons showed close identity of California and Arizona isolates with 2005 Argentine isolates, suggesting introduction from South America and underscoring the value of continued arbovirus surveillance.

Evolutionary genomics of Culex pipiens: global and local adaptations associated with climate, life-history traits and anthropogenic factors.

We present the first genome-wide study of recent evolution in Culex pipiens species complex focusing on the genomic extent, functional targets and likely causes of global and local adaptations. We resequenced pooled samples of six populations of C. pipiens and two populations of the outgroup Culex torrentium. We used principal component analysis to systematically study differential natural selection across populations and developed a phylogenetic scanning method to analyse admixture without haplotype data. We found evidence for the prominent role of geographical distribution in shaping population structure and specifying patterns of genomic selection. Multiple adaptive events, involving genes implicated with autogeny, diapause and insecticide resistance were limited to specific populations. We estimate that about 5-20% of the genes (including several histone genes) and almost half of the annotated pathways were undergoing selective sweeps in each population. The high occurrence of sweeps in non-genic regions and in chromatin remodelling genes indicated the adaptive importance of gene expression changes. We hypothesize that global adaptive processes in the C. pipiens complex are potentially associated with South to North range expansion, requiring adjustments in chromatin conformation. Strong local signature of adaptation and emergence of hybrid bridge vectors necessitate genomic assessment of populations before specifying control agents.

Extrinsic Incubation Rate is Not Accelerated in Recent California Strains of West Nile Virus in Culex tarsalis (Diptera: Culicidae).

The efficiency of West Nile virus (WNV) transmission by competent mosquito vectors is driven by temperature and defined, in part, by the extrinsic incubation period, which is the time from a mosquito's consumption of an infected bloodmeal until it becomes capable of transmitting the virus to the next vertebrate host. The extrinsic incubation period can be altered by a variety of factors involved in vector-pathogen interactions, and in North America, the WN02 strain of WNV emerged and displaced the founding NY99 strain reportedly because the duration of the extrinsic incubation period in Culex mosquitoes was shortened by a single positively selected mutation. However, recent work has suggested that this change is not universal and may depend on vector species or strain. In the current study, we estimated the extrinsic incubation periods at 22 and 30°C in Culex tarsalis Coquillett. We found that the time to transmission of the original North American WNV strain, NY99, was not different from two more recent California isolates of the WN02 genotype: one of the earliest California isolates from the southeastern deserts, and a more recent 2011 isolate from a hyperendemic region in the Central Valley. We conclude with a model-based assessment of the epidemiological effects of temperature on the duration of mosquitoes' infectious life, which estimated that most mosquitoes have an infectious life of only a few days, but its duration expands markedly at warmer temperatures.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

West Nile virus and non-West Nile virus mortality and coinfection of American crows (Corvus brachyrhynchos) in California.

American crows are acutely sensitive to West Nile virus (WNV) infection, and crow mortality has been used in WNV surveillance to monitor enzootic transmission. However, non-WNV sources of mortality could reduce the reliability of crow death as a surveillance tool. Here, using a combination of histopathologic, toxicologic, virologic, and molecular techniques we describe causes of mortality in 67 American crows (Corvus brachyrhynchos) that were collected from a population in the Sacramento Valley of California in 2012 and 2013. Evidence of infectious disease was detected in 70% (47/67) of carcasses. The majority of deaths were linked to a suite of non-WNV viral, bacterial, and fungal infections (39%; 23/59 cases), WNV (36%; 24/67 cases), and an acute toxic event (25%; 15/59 cases). Coinfections were detected in 20% (12/59) of birds and frequently were associated with WNV and poxviral dermatitis. Inferences about WNV activity based on crow mortality should be supported by laboratory confirmation because crow mortality frequently can be caused by other infectious diseases or toxic events.

Host selection patterns of Culex tarsalis (Diptera: Culicidae) at wetlands near the Salton Sea, Coachella Valley, California, 1998-2002.

The bloodmeal hosts used by Culex tarsalis Coquillett collected along the Salton Sea in Coachella Valley, CA, during 1998-2002 were identified using sequences of the cytochrome c oxidase I gene identified from Barcode of Life database. Overall, 265 (83.3%) of 318 bloodmeals were identified, of which 76.6% fed on birds, 18.1% on mammals, and 5.3% on reptiles. Forty-seven different hosts were identified, none of which comprised > 12.5% of the total. Although Cx. tarsalis exhibits specific host-seeking flight patterns, bloodmeals seemed to be acquired opportunistically, thereby limiting potential arbovirus transmission efficiency in species-rich environments.

Host-selection patterns of Culex tarsalis (Diptera: Culicidae) determine the spatial heterogeneity of West Nile virus enzootic activity in northern California.

The spatial heterogeneity of West Nile virus (WNV) activity in Sutter County, CA, as measured by mosquito infection rates, was associated with spatial variation in the prevalence of Culex blood feeding on competent passeriform hosts. Overall, 42 vertebrate host species (31 avian, 11 mammal) were identified from 601 blood-fed Culex tarsalis Coquillett and 151 blood-fed Culex pipiens L. complex females using sequences of the cytochrome c oxidase I gene and the Barcode of Life Data Systems database. WNV infection rates were low at sites where the primary vector, Cx. tarsalis, fed frequently on domestic cattle or incompetent galliform birds and high when females fed frequently on American Robins, American Crows, and Yellow-billed Magpies. Opportunistic host selection by Cx. tarsalis in combination with spatial variation in the presence of highly competent corvid hosts appeared to determine the distribution of WNV activity in rural Sutter County, CA.

Experimental and natural vertical transmission of West Nile virus by California Culex (Diptera: Culicidae) mosquitoes.

Culex (Diptera: Culicidae) mosquitoes, the primary summer vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), also may serve as overwintering reservoir hosts. Detection of WN viral RNA from larvae hatched from eggs deposited by infected females during late summer and fall may provide evidence for the vertical passage of WNV to overwintering cohorts. To determine whether vertical transmission to the overwintering generation occurs in populations of Culex mosquitoes throughout California, larvae from naturally infected females were tested by family for WN viral RNA by real-time quantitative reverse transcription-polymerase chain reaction during August through October 2011. Viral RNA was detected in 34 of 934 Culex tarsalis Coquillett and Cx. pipiens complex females that laid viable egg rafts. From these egg rafts, first-instar larvae from nine families tested positive, yielding an overall field vertical transmission rate of 26% (n = 34). To determine whether the WNV may be lost transtadially during development to the adult stage, first-instar larvae and adult progeny from experimentally infected Cx. pipiens complex females were assessed for the presence and quantity of WN viral RNA. Most (approximately 75%) WNV infections were lost from positive families during larval development to the adult stage. In field and laboratory studies, only infected mothers with mean cycle threshold scores < or = 20 vertically transmitted WNV to larval progeny, adult progeny, or both. In summary, vertical transmission of WNV was detected repeatedly in naturally infected Culex mosquitoes collected throughout California during late summer and fall, with females having high titered infections capable of passing WNV onto their progeny destined for overwintering.

Overwintering biology of Culex (Diptera: Culicidae) mosquitoes in the Sacramento Valley of California.

At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010-2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to >or=stage I-II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but host-seeking seemed limited by temperature. An accrual of 97.52 +/- 30.7 and 162.85 +/- 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.

Epidemic versus endemic West Nile virus dead bird surveillance in California: Changes in sensitivity and focus.

Since 2003, the California West Nile virus (WNV) dead bird surveillance program (DBSP) has monitored publicly reported dead birds for WNV surveillance and response. In the current paper, we compared DBSP data from early epidemic years (2004-2006) with recent endemic years (2018-2020), with a focus on specimen collection criteria, county report incidence, bird species selection, WNV prevalence in dead birds, and utility of the DBSP as an early environmental indicator of WNV. Although fewer agencies collected dead birds in recent years, most vector control agencies with consistent WNV activity continued to use dead birds as a surveillance tool, with streamlined operations enhancing efficiency. The number of dead bird reports was approximately ten times greater during 2004-2006 compared to 2018-2020, with reports from the Central Valley and portions of Southern California decreasing substantially in recent years; reports from the San Francisco Bay Area decreased less dramatically. Seven of ten counties with high numbers of dead bird reports were also high human WNV case burden areas. Dead corvid, sparrow, and quail reports decreased the most compared to other bird species reports. West Nile virus positive dead birds were the most frequent first indicators of WNV activity by county in 2004-2006, followed by positive mosquitoes; in contrast, during 2018-2020 mosquitoes were the most frequent first indicators followed by dead birds, and initial environmental WNV detections occurred later in the season during 2018-2020. Evidence for WNV impacts on avian populations and susceptibility are discussed. Although patterns of dead bird reports and WNV prevalence in tested dead birds have changed, dead birds have endured as a useful element within our multi-faceted WNV surveillance program.

Comparison of enzootic risk measures for predicting West Nile disease, Los Angeles, California, USA, 2004-2010.

In Los Angeles, California, USA, 2 epidemics of West Nile virus (WNV) disease have occurred since WNV was recognized in 2003. To assess which measure of risk was most predictive of human cases, we compared 3 measures: the California Mosquito-Borne Virus Surveillance and Response Plan Assessment, the vector index, and the Dynamic Continuous-Area Space-Time system. A case-crossover study was performed by using symptom onset dates from 384 persons with WNV infection to determine their relative environmental exposure to high-risk conditions as measured by each method. Receiver-operating characteristic plots determined thresholds for each model, and the area under the curve was used to compare methods. We found that the best risk assessment model for human WNV cases included surveillance data from avian, mosquito, and climate sources.

Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes.

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.

Evaluating sampling method bias in Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae) bloodmeal identification studies.

Determining the bloodmeal hosts of the Culex vectors of encephalitis viruses such as West Nile virus is essential for understanding the role of these mosquitoes in enzootic and epidemic transmission. Although molecular techniques have increased our knowledge of blood feeding patterns by allowing host identification to the species level, few studies have focused on the role that sampling methods may play in determining these patterns. In the current study, we identified 644 bloodmeals from Culex tarsalis Coquillett and Culex quinquefasciatus Say females collected in CO2 traps (dry ice-baited Center for Disease Control traps), in gravid traps, and aspirated from resting sites. There was no significant difference in the bloodmeal host apportionment in sampling methods such as gravid traps and resting collections that collected fully engorged females. However, CO2 traps that collected partially fed females had a significantly different apportionment of hosts than either gravid or resting collections. Bloodfed females from CO2 traps had either fed on only a small subset of available host species or were biased toward more mammalian and fewer nonpasserine avian feeds than females from other collections. Because both full and partial bloodmeals can contribute to viral transmission, obtaining Culex bloodmeal collections from multiple sampling methods may be important to fully interpret the role of these mosquitoes as maintenance and/or bridge vectors.

Use of scented sugar bait stations to track mosquito-borne arbovirus transmission in California.

Laboratory and field research was conducted to determine if Culex tarsalis Coquillett expectorated West Nile virus (WNV) during sugar feeding and if a lure or bait station could be developed to exploit this behavior for WNV surveillance. Experimentally infected Cx. tarsalis repeatedly expectorated WNV onto filter paper strips and into vials with wicks containing sucrose that was readily detectable by a quantitative reverse transcriptase-polymerase chain reaction assay. Few females (33%, n = 27) became infected by imbibing sugar solutions spiked with high concentrations (10(7) plaque forming units/ml) of WNV, indicating sugar feeding stations probably would not be a source of WNV infection. In nature, sugar bait stations scented with the floral attractant phenyl acetaldehyde tracked WNV transmission activity in desert but not urban or agricultural landscapes in California. When deployed in areas of the Coachella Valley with WNV activity during the summer of 2011, 27 of 400 weekly sugar samples (6.8%) tested positive for WNV RNA by reverse transcriptase-polymerase chain reaction. Prevalence of positives varied spatially, but positive sugar stations were detected before concurrent surveillance measures of infection (mosquito pools) or transmission (sentinel chicken seroconversions). In contrast, sugar bait stations deployed in urban settings in Los Angeles or agricultural habits near Bakersfield in Kern County supporting WNV activity produced 1 of 90 and 0 of 60 positive weekly sugar samples, respectively. These results with sugar bait stations will require additional research to enhance bait attractancy and to understand the relationship between positive sugar stations and standard metrics of arbovirus surveillance.

Importance of recrudescent avian infection in West Nile virus overwintering: incomplete antibody neutralization of virus allows infrequent vector infection.

After the acute infection period, birds persistently infected with West Nile virus (family Flaviviridae, genus Flavivirus, WNV) occasionally shed virus into the bloodstream, but these virions normally are inactivated by neutralizing antibody. The current work tested the hypothesis that these host neutralizing antibodies protect mosquito vectors from WNV infection and reevaluated the minimum WNV infectious dose necessary to infect Culex tarsalis Coquillett. To determine whether host antibodies protect mosquitoes from infection, Cx. tarsalis and Culex stigmatosoma Dyar were fed bloodmeals containing avian blood, WNV, and sera with or without WNV-specific neutralizing antibodies. When viral particles were completely bound by antibody, mosquitoes were protected from infection; however, when incompletely bound, WNV titers as low as 10(2.3) plaque-forming units (pfu)/ml resulted in 5% infection. These data indicated that avian antibodies were protective to mosquito vectors and were not dissociated during digestion. Because recrudescent viremias may not attain the same magnitude as initial acute viremias, Cx. tarsalis vector competence was reevaluated focusing on the fate of low-titered bloodmeals. Females were evaluated for vector competence after ingesting bloodmeals containing 10(2.2), 10(3.4), 10(4.5), 10(5.5), or 10(6.5) WNV pfu/ml. Infection increased with bloodmeal titer, with 1% of the mosquitoes ingesting 10(3.4) pfu/ml and 45% of the mosquitoes ingesting 10(6.5) pfu/ml developing disseminated infections. The incomplete neutralization of recrudescent virus may be sufficient to infect a low proportion of competent blood-feeding Culex mosquitoes and perhaps allow persistently infected birds to provide a mechanism for arbovirus overwintering.

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.

The contrasting bionomics of Culex mosquitoes in western North America.

Mosquitoes in the genus Culex are the primary enzootic maintenance and bridge vectors of the North American encephalitides, now including West Nile virus. This review briefly summarizes the biology of three key vector species in western North America, Culex tarsalis, Cx. pipiens complex and Cx. stigmatosoma, focusing on the long history of research done in California. Topics reviewed include population genetic structure, larval ecology, autogeny, mating behavior, host-seeking behavior, host-selection patterns, and overwintering strategies. These attributes collectively have allowed the successful exploitation of anthropogenically altered ecosystems and enabled the role of these species as maintenance and bridge vectors of arboviruses.