Bird species define the relationship between West Nile viremia and infectiousness to <i>Culex pipiens</i> mosquitoes

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Abstract

The transmission cycle of West Nile virus (WNV) involves multiple species of birds. The relative importance of various bird species to the overall transmission is often inferred from the level and duration of viremia that they experience upon infection. Reports utilizing in vitro feeding techniques suggest that the source and condition of blood in which arboviruses are fed to mosquitoes can significantly alter the infectiousness of arbovirus to mosquitoes. We confirmed this using live hosts. A series of mosquito feedings with Culex pipiens was conducted on WNV-infected American robins and common grackles over a range of viremias. Mosquitoes were assayed individually by plaque assay for WNV at 3 to 7 days after feeding. At equivalent viremia, robins always infected more mosquitoes than did grackles. We conclude that the infectiousness of viremic birds cannot always be deduced from viremia alone. If information concerning the infectiousness of a particular bird species is important, such information is best acquired by feeding mosquitoes directly on experimentally infected individuals of that species.

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We injected West Nile virus into seronegative individuals of two bird species, American Robin and Common Grackle, and then fed Culex pipiens mosquitoes on these birds during the viremic period. We found that, despite having equivalent or lower levels of viremia, robins infected significantly more mosquitoes than did grackles. The reasons for this are not known, but our results indicate that the relationship between host viremia and infectiousness is not always straight forward and may vary among different host species. To gain precise information for a mosquito-borne arbovirus system, it is best to conduct mosquito feedings on live viremic hosts rather than extrapolate the infectiousness of a host species based solely on its viremia profile.

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The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations.

Woolf Walker, Francesca D. Frentiu, Conor McMeniman … (2011)

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

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West Nile Virus Epidemics in North America Are Driven by Shifts in Mosquito Feeding Behavior

A. Kilpatrick, Laura D. Kramer, Matthew J. Jones … (2006)

Introduction Approximately three quarters of human emerging infectious diseases are caused by zoonotic pathogens [ 1]. For many of these diseases, nonhuman animals are the primary host and human infections are incidental and often dead-end in nature [ 1]. Nonetheless, these pathogens can have severe consequences for human health, due to high mortality rates, high incidence rates, or both [ 1– 4]. The factors that drive the spillover of these pathogens from other animals to humans determine the intensity of human epidemics [ 5]. For directly transmitted zoonotic pathogens, transmission to humans increases with the contact rate between infected animals and susceptible humans, whereas for vector-borne diseases, the feeding behavior and feeding preferences of the vector(s) play a key role in determining the force of infection [ 6]. Therefore, a critical step in the control of epidemics of zoonotic vector-borne pathogens is determining the feeding preferences of key vectors and how they change over space and time [ 5]. West Nile virus (WNV) is a zoonotic pathogen that is maintained in an enzootic cycle primarily through transmission between viremic birds and ornithophilic (bird-biting) mosquitoes [ 7]. However, during late summer and early fall, WNV also infects humans and other mammals and has caused repeated large-scale human epidemics in North America since it was first detected in New York City in 1999 [ 8]. It has caused over 20,000 reported cases, 770 deaths, and an estimated 215,000 illnesses during the past 7 y and is now the dominant vector-borne disease in North America [ 9– 11]. During the past 4 y, there were epidemics in North America of approximately 2,000 to 10,000 cases each, far larger than European epidemics, which have never exceeded 400 cases and occur infrequently [ 12]. Three hypotheses have been put forth to explain the increased intensity of epidemics in North America. First, the strain of WNV introduced in North America has been shown to be more virulent to American crows (Corvus brachyrhynchos) than one strain circulating in the Old World [ 13]. Second, North American birds had no previous exposure to WNV and the lack of acquired and evolved immunity may have increased the intensity of epidemics [ 14]. Third, North American Culex pipiens mosquitoes, the dominant vector of WNV in the northeast and north-central United States, appear to be hybrids of the bird-biting “pipiens” form and human-feeding “molestus” form of Old World Cx. pipiens [ 15]. These three hypotheses are neither mutually exclusive nor exhaustive. We tested a fourth hypothesis, that epidemics are driven by a shift in mosquito feeding behavior from birds to mammals, as has been demonstrated for other mosquitoes in North America [ 16– 18] but not for Cx. pipiens in the New or Old World [ 19]. The most competent vertebrate hosts for WNV appear to be birds [ 20], whereas most mammals, including humans, are poorly competent hosts [ 21, 22]. If mosquitoes feed primarily on birds in the summer and then switch to humans in the fall, this may amplify both the intensity of the summer epidemic in mosquitoes and birds and later transmission of WNV to humans. To test this hypothesis and to understand the factors driving North American human WNV epidemics, we integrated data on the feeding behavior, population dynamics, and epidemiology of mosquitoes, birds, and humans in three regions across the United States. Results PCR and DNA Sequencing of Mosquito Blood Meals The probability that Cx. pipiens fed on humans increased over the mosquito season ( Figure 1A; logistic regression of individual blood meals on Julian date: intercept = −7.3, slope = 0.025; n = 148 blood meals; p = 0.004). The probability that Cx. pipiens fed on mammals also increased ( Figure 1A; intercept = −6.8, slope = 0.025; n = 148 blood meals; p < 0.001). However, the probability of feeding on nonhuman mammals did not increase significantly (intercept = −6.5, slope = 0.017; n = 148 blood meals; p = 0.14), suggesting that the increase in mosquitoes' preference for humans was responsible for the apparent shift to mammals. As would be expected, the probability of feeding on avian hosts declined over the same period (intercept = 11.9, slope = −0.046; n = 148; p < 0.001). Using the fitted logistic equations, the probability of Cx. pipiens feeding on humans and mammals increased from 0.040 and 0.064 in mid-June to 0.28 and 0.39 in mid-September, which roughly match the monthly groupings of the data ( Figure 1A). These are 7-fold and 6-fold increases, respectively. Changes in Mosquitoes' Preferred Host In early summer (May and June), 51% of Cx. pipiens' blood meals came from a widespread, competent avian host, the American robin (Turdus migratorius) [ 20, 23], despite the fact that this species made up only 4.5% of the avian community ( Figure 1B) (2% to 7% at each site). The abundance of robins in urbanized areas declined over the next 4 mo ( Figure 1B; relative abundance of American robins = 3.25 − 0.0116 * Julian date; n = 24; R 2 = 30.2%; p = 0.004), as robins dispersed after breeding. As the availability of robins declined, the probability they were fed upon by Cx. pipiens also declined markedly ( Figure 1B; logistic regression of individual blood meals on Julian date: intercept = 6.5, slope = −0.037; n = 148; p < 0.001), which coincided with the rise of humans as an important source of Cx. pipiens blood meals ( Figure 1A). In contrast, the total abundance of birds, which was dominated by house sparrows, Passer domesticus (42% to 67% of total abundance), increased over this period as a result of reproduction ( Figure 1B). Thus, the shift in Cx. pipiens feeding from birds to humans is not a result of decreasing avian abundance but is more likely the result of the decline in the abundance of robins, their preferred host. Predicted and Actual Human WNV Infections Our model-predicted risk of the transmission of WNV to humans by Cx. pipiens peaked from late July to mid-August, declined in late August, and increased slightly at the end of September ( Figure 1C) before a period of cold weather greatly decreased mosquito activity in early October. Human WNV infections in the surrounding area showed a strikingly similar pattern ( Figure 1C), with a steep rise in cases occurring approximately 14 d after the peak in predicted risk, in agreement with the 3- to 14-d period between infection and the onset of illness in humans [ 24] and the approximately 7-d delay between becoming infected and becoming infectious when a mosquito is initially infected with WNV [ 25]. The correlation of our predicted risk and human cases 14 d later was highly significant ( r = 0.80; n = 8 biweekly accumulations from mid-June through October; p = 0.009). Finally, our data-driven model suggests that had mosquitoes continued to feed on humans with the same probability that they did in June, human epidemics would have occurred at much lower intensity and there would have been few, if any, infections after mid-September ( Figure 1C: Human infection risk [no shift]). Feeding Shifts and WNV Epidemics in Other Parts of North America Previously documented shifts from birds to mammals in Cx. tarsalis in California [ 18] and Colorado [ 17] also intensify WNV epidemics in these states ( Figure 2). The fraction of feedings from mammals in peak WNV months (July through September) in California and Colorado were 4.1- and 1.7-fold higher, respectively, than those in June, suggesting that feeding shifts substantially increased the number of human WNV cases in these regions. The July and August peaks in human WNV cases in Colorado and California follow the peak in the abundance of WNV-infected mosquitoes that occur in June and July, respectively [ 26, 27], and are amplified and extended by the shift in Cx. tarsalis feeding from birds to mammals. These feeding shifts occur during the late summer periods of postbreeding dispersal and migration of robins in these regions ( Figure 2). In support of the link between feeding patterns and the dispersal and decline of passerine migrants, the fraction of feedings of Cx. tarsalis from passerines (many of which are competent for WNV [ 20, 28]) decreased from 40.5 ± 1.9% of feedings in May through June to 20.0 ± 1.0% in July through September in Colorado (correlation with time: r = −0.90; n = 5 mo; p = 0.037; data from [ 17]). Discussion Our data from the mid-Atlantic demonstrate a late-summer shift in the feeding behavior of Cx. pipiens from their preferred avian host, American robins, to humans. This shift offers an explanation for the late-summer timing and increased intensity of human WNV epidemics in the northeast and north-central parts of North America [ 11, 29], where this species is the dominant enzootic vector and bridge vector [ 30]. We have also shown that WNV epidemics in the central and western parts of the United States are intensified by feeding shifts that escalate as robins and other WNV-competent avian hosts disperse and migrate following breeding. Finally, an important WNV vector in the southeast United States, Cx. nigripalpus [ 31], also shifts from feeding primarily on birds in spring to an increasing fraction from mammals in later summer, although the cause for this shift is unknown [ 16]. Taken together, these data suggest that mosquito feeding shifts appear to be a continent-wide phenomenon that amplifies the transmission of WNV from birds to humans and other mammals. Feeding shifts have two synergistic effects on the intensity of WNV transmission to humans. First, we have shown that the increase over time in the probability of Cx. pipiens feeding on humans results in a greater number of human WNV infections than if the mosquitoes fed on humans with the same probability as in early summer ( Figure 1C). Second, feeding primarily on WNV-competent avian hosts during the amplification period of the epidemiological cycle maximizes the intensity of the epidemic in mosquitoes. If mosquitoes fed on humans in early summer with the same probability as that seen in late summer (approximately 0.30), the peak and mean prevalence of WNV in mosquitoes would be substantially reduced [ 32, 33], due to the large number of wasted feedings on humans, which, like immune birds, are dead-end hosts [ 21]. In contrast, holding the probability of mosquitoes feeding on humans at its lowest level, 0.04 (as we modeled in Figure 1C), has only a minor effect on WNV amplification. This is because mosquito WNV prevalences are already beginning to decline (possibly as a result of increased acquired immunity in juvenile birds) when mammals begin to make up an important fraction of the blood meals. Thus, the shift in feeding from competent hosts early in the season to humans later leads first to greater amplification of the virus as transmission intensifies between birds and mosquitoes and subsequently to an even greater number of human WNV infections. Feeding shifts from birds to humans offer a broader and more direct explanation for the intensity of North American WNV epidemics compared to those in Europe than those previously proposed: hybridization of the “pipiens” and “molestus” forms of Cx. pipiens [ 15], the novelty of WNV for North American birds [ 14], and the virulence of the introduced strain compared to an Old World isolate [ 13]. First, although it is possible that the feeding shift we observed in Cx. pipiens could be the result of increasing hybridization between the molestus and pipiens forms of Cx. pipiens [ 15], this has yet to be demonstrated, and a shift due to robin dispersal is a more parsimonious explanation. In any case, increasing hybridization would offer an explanation only in areas where Cx. pipiens is the dominant vector. In addition, a temporally invariant increase in feeding on humans by hybrid Cx. pipiens as proposed [ 15, 19] would have counteracting effects on mosquito and human WNV infections, as we have shown. Second, WNV epizootics occur in birds after the production of large numbers of naïve and susceptible offspring, so the acquired immunity of adults may play only a limited role in subsequent transmission. Third, the introduced strain of WNV appears to be more virulent compared to an Old World strain in American crows (Corvus brachyrhynchos) [ 13], but not in more important amplification hosts such as house sparrows (Passer domesticus) [ 34, 35]. Finally, high vector competence of Cx. tarsalis [ 36] and increases in the abundance or WNV infection prevalence of more mammalophilic mosquito species may also contribute to WNV transmission to humans. However, the latter appears to be of limited importance in the northeast United States where the enzootic vectors appear to be responsible for more than 80% of human WNV infections [ 30]. In sum, although many factors influence WNV transmission to humans, our results suggest that feeding shifts occur across North America and offer a direct explanation for the greater number of human WNV infections compared to the Old World. More broadly, our findings show that shifts in mosquito feeding behavior are a geographically widespread phenomenon that intensifies epidemics of several avian zoonotic viruses including WNV, Western equine encephalitis virus, and St. Louis encephalitis virus [ 16] , and may affect other zoonotic vector-borne pathogens. Studies aimed at determining the factors that influence pathogen transmission to humans are critical to reducing the impact of these and other devastating diseases. Materials and Methods We collected field data at six sites in Maryland and Washington, D. C., from May through September 2004. The sites included three urban areas, the National Mall in Washington, D. C., northeast of the Watergate Hotel in Foggy Bottom, D. C., and west of Camden Yards in Baltimore, Maryland; two residential areas, Takoma Park, Maryland, and Bethesda, Maryland; and one park within an urban setting, Fort Dupont Park, D. C. Each site was approximately 1 km in diameter, with a relatively homogeneous percent forest cover and land use in the site and surrounding area. The sites span a distance of greater than 40 miles and are separated by 3 to 25 miles. We estimated the abundance of host-seeking Culex mosquitoes at each site using eight CDC light traps, baited with dry ice (CO 2). We ran these traps for two nights every 2 wk from May to September at each site and averaged abundance across the sites. To determine the species composition of Culex mosquitoes at our sites, which are difficult to identify morphologically [ 37], we used PCR on the internal spacer gene [ 38] to identify all engorged Culex mosquitoes to species and an additional subset of 40 Culex mosquitoes at each site. We found that more than 90% of the Culex mosquitoes identified at each of the six sites were Cx. pipiens. We collected engorged mosquitoes from each site using CDC light and gravid traps and by collecting mosquitoes resting on vegetation with a large backpack-mounted aspirator. Blood meals were identified as avian, mammalian, or other following extraction of DNA from the engorged mosquitoes and PCR amplification of the cytochrome b gene as described and verified [ 39]. Hosts were identified to the species level by nucleotide sequencing of the amplified product using an ABI 3700 DNA Analyzer (Applied Biosystems, Foster City, California, United States) with avian or mammalian forward and reverse primers [ 39]. Data were analyzed using the DNAStar software package (Madison, Wisconsin, United States) and sequence analysis was conducted using Blastn via the Internet. Only engorged mosquitoes identified as Cx. pipiens were used in the feeding analysis. We obtained PCR product for 165 blood meals and DNA sequences for 148 of these. Mosquitoes were tested for WNV RNA using real-time RT-PCR [ 40] in groups (pools) of 20 to 50 individuals. The WNV infection prevalence was estimated using maximum likelihood techniques [ 41] and expressed as the WNV infection rate = 1,000 × mosquito WNV infection prevalence. We modeled the risk of the transmission of WNV to humans by Cx. pipiens as the product of Culex mosquito abundance (more than 90% of which were Cx. pipiens), Culex WNV infection prevalence, and probability of feeding on humans [ 30]. We calculated the variance of predicted risk using a Taylor series expansion approximation: where the x i parameters represent the n = 3 parameters in the risk model. We compared predicted WNV risk to the number of human cases in Maryland [ 42, 43]. We corrected the date of reporting by obtaining the date of the onset of illness for each case (unpublished data, A. Bergmann, Maryland Department of Health and Mental Hygiene). We estimated the abundance of birds using four- to six-point transects, six minutes in duration, performed at each site monthly from May through September and analyzed the data using Program Distance [ 44], which accounts for species' differences in detectability. We estimated the decline of American robin abundance ( Figure 1B) over time using a regression of the relative abundance of robins at each site on the Julian date. The changes in robin and total bird abundance were similar across the six sites. To examine the temporal overlap between feeding shifts and human WNV epidemics in other parts of North America, we obtained data on temporal variation in feeding on birds and mammals for Cx. tarsalis in Colorado [ 17] and California [ 18]. We compared these patterns with data on the date of onset of illness and number of human cases of WNV in Colorado [ 45] and California [ 27] in 2004. Finally, we compared these to periods of postbreeding dispersal and migration for robins in California [ 23, 46] and in four different habitats in Arizona [ 47– 49].

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A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood.

Olof Hellgren, Jonas Waldenström, Staffan Bensch (2004)

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).

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Contributors

Jefferson A. Vaughan:

ORCID: https://orcid.org/0000-0002-7067-0654

Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draftRole: Writing – review & editing

Robert A. Newman: Role: Formal analysisRole: Writing – review & editing

Michael J. Turell: Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing

Duane J. Gubler: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Negl Trop Dis

Journal ID (iso-abbrev): PLoS Negl Trop Dis

Journal ID (publisher-id): plos

Title: PLoS Neglected Tropical Diseases

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Print): 1935-2727

ISSN (Electronic): 1935-2735

Publication date (Electronic): 6 October 2022

Publication date Collection: October 2022

Volume: 16

Issue: 10

Electronic Location Identifier: e0010835

Affiliations

[1 ] Department of Biology, University of North Dakota, Grand Forks, North Dakota, United States of America

[2 ] Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America

Duke-NUS GMS, SINGAPORE

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The authors have declared that no competing interests exist.

[¤]

Current address: VectorID LLC, Frederick, Maryland, United States of America

* E-mail: jefferson.vaughan@ 123456und.edu

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Jefferson A. Vaughan https://orcid.org/0000-0002-7067-0654

Article

Publisher ID: PNTD-D-22-00658

DOI: 10.1371/journal.pntd.0010835

PMC ID: 9578590

PubMed ID: 36201566

SO-VID: 77a45f73-bcc1-4e4f-80a5-5d4420262931

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This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

History

Date received : 19 May 2022

Date accepted : 20 September 2022

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Figures: 2, Tables: 3, Pages: 13

Funding

Funded by: funder-id http://dx.doi.org/10.13039/100015691, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases;

Award ID: R21 AI105662

Award Recipient :

ORCID: https://orcid.org/0000-0002-7067-0654

Jefferson A. Vaughan

This study was funded by a grant from the National Institute of Allergy & Infectious Diseases ( https://www.niaid.nih.gov, Award R21 AI105662 to J.A.V.) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Publication Update 2022-10-18

Data Availability All relevant data are within the manuscript and its Supporting information files.

Bird species define the relationship between West Nile viremia and infectiousness to Culex pipiens mosquitoes

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The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations.

West Nile Virus Epidemics in North America Are Driven by Shifts in Mosquito Feeding Behavior

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood.

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