Xenosurveillance reflects traditional sampling techniques for the identification of human pathogens: A comparative study in West Africa

BACKGROUND: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. METHODOLOGY/PRINCIPAL FINDINGS: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. CONCLUSIONS/SIGNIFICANCE: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens

A positively selected mutation in the WNV 2K peptide confers resistance to superinfection exclusion in vivo

AbstractMolecular epidemiologic studies of North American (NA) West Nile virus (WNV; Flaviviridae, Flavivirus) have documented the displacement of the introduced NY99 genotype with WN02. In addition, these studies have shown that particular substitutions are under positive selection. One occurs in the C-terminus of the NS4A coding sequence and results in a valine to methionine substitution at position nine of the 2K peptide. 2K-V9M confers the ability to overcome superinfection exclusion in vitro. We hypothesized that WNV strains bearing 2K-V9M have higher fitness than wildtype in Culex quinquefasciatus mosquitoes. Although infection rates and viral titers were not significantly different, virus dissemination rates were significantly higher with WNV 2K-V9M. As a super-infecting virus, WNV 2K-V9M was more successful than wildtype, however, in a mixed infection, 2K-V9M was not. These data support observations that 2K-V9M confers a context-specific selective advantage in mosquitoes and provides an in vivo mechanism for its positive selection

Crow Deaths Caused by West Nile Virus during Winter

In New York, an epizootic of American crow (Corvus brachyrhynchos) deaths from West Nile virus (WNV) infection occurred during winter 2004–2005, a cold season when mosquitoes are not active. Detection of WNV in feces collected at the roost suggests lateral transmission through contact or fecal contamination

Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense

<p>Abstract</p> <p>Background</p> <p>Small RNA (sRNA) regulatory pathways (SRRPs) are important to anti-viral defence in mosquitoes. To identify critical features of the virus infection process in Dengue serotype 2 (DENV2)-infected <it>Ae. aegypti</it>, we deep-sequenced small non-coding RNAs. Triplicate biological replicates were used so that rigorous statistical metrics could be applied.</p> <p>Results</p> <p>In addition to virus-derived siRNAs (20-23 nts) previously reported for other arbovirus-infected mosquitoes, we show that PIWI pathway sRNAs (piRNAs) (24-30 nts) and unusually small RNAs (usRNAs) (13-19 nts) are produced in DENV-infected mosquitoes. We demonstrate that a major catalytic enzyme of the siRNA pathway, Argonaute 2 (Ago2), co-migrates with a ~1 megadalton complex in adults prior to bloodfeeding. sRNAs were cloned and sequenced from Ago2 immunoprecipitations. Viral sRNA patterns change over the course of infection. Host sRNAs were mapped to the published aedine transcriptome and subjected to analysis using edgeR (Bioconductor). We found that sRNA profiles are altered early in DENV2 infection, and mRNA targets from mitochondrial, transcription/translation, and transport functional categories are affected. Moreover, small non-coding RNAs (ncRNAs), such as tRNAs, spliceosomal U RNAs, and snoRNAs are highly enriched in DENV-infected samples at 2 and 4 dpi.</p> <p>Conclusions</p> <p>These data implicate the PIWI pathway in anti-viral defense. Changes to host sRNA profiles indicate that specific cellular processes are affected during DENV infection, such as mitochondrial function and ncRNA levels. Together, these data provide important progress in understanding the DENV2 infection process in <it>Ae. aegypti</it>.</p

Detection by Enzyme-Linked Immunosorbent Assay of Antibodies to West Nile virus in Birds

We adapted an indirect immunoglobulin G enzyme-linked immunosorbent assay to facilitate studies of West Nile virus (WNV) and evaluated its application to taxonomically diverse avian species. Anti-WNV antibodies were detected in 23 bird species, including many exotic species, demonstrating its value in studies of WNV epizootiology

Impact of Simultaneous Exposure to Arboviruses on Infection and Transmission by Aedes aegypti Mosquitoes

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection

SARS‑CoV‑2 entry into and evolution within a skilled nursing facility

SARS-CoV-2 belongs to the family Coronaviridae which includes multiple human pathogens that have an outsized impact on aging populations. As a novel human pathogen, SARS-CoV-2 is undergoing continuous adaptation to this new host species and there is evidence of this throughout the scientific and public literature. However, most investigations of SARS-CoV-2 evolution have focused on largescale collections of data across diverse populations and/or living environments. Here we investigate SARS-CoV-2 evolution in epidemiologically linked individuals within a single outbreak at a skilled nursing facility beginning with initial introduction of the pathogen. The data demonstrate that SARSCoV- 2 was introduced to the facility multiple times without establishing an interfacility transmission chain, followed by a single introduction that infected many individuals within a week. This largescale introduction by a single genotype then persisted in the facility. SARS-CoV-2 sequences were investigated at both the consensus and intra-host variation levels. Understanding the variability in SARS-CoV-2 during transmission chains will assist in understanding the spread of this disease and can ultimately inform best practices for mitigation strategies

The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice

AbstractTo define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype

Impact of Extrinsic Incubation Temperature on Natural Selection During Zika Virus Infection of Aedes Aegypti and Aedes Albopictus

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of \u3e 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes