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

Aedes aegypti uses RNA interference in defense against Sindbis virus infection

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an important anti-viral defense mechanism. The <it>Aedes aegypti </it>genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use <it>Ae. aegypti </it>as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family <it>Togaviridae</it>, genus <it>Alphavirus</it>).</p> <p>Results</p> <p>SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate <it>Ago2</it>, <it>Dcr2</it>, or <it>TSN </it>mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut <it>Ago2</it>, <it>Dcr2</it>, and <it>TSN </it>transcript levels during infection revealed that only <it>TSN </it>transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.</p> <p>Conclusion</p> <p>We show that silencing RNAi components in <it>Ae. aegypti </it>results in transient increases in SINV replication. Furthermore, <it>Ae. aegypti </it>RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in <it>Ae. aegypti</it>.</p

Mosquitoes Transmit Unique West Nile Virus Populations During Each Feeding Episode

Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level

West African Anopheles Gambiae Mosquitoes Harbor a Taxonomically Diverse Virome Including New Insect-Specific Flaviviruses, Mononegaviruses, and Totiviruses

Anopheles gambiae are a major vector of malaria in sub-Saharan Africa. Viruses that naturally infect these mosquitoes may impact their physiology and ability to transmit pathogens. We therefore used metagenomics sequencing to search for viruses in adult Anopheles mosquitoes collected from Liberia, Senegal, and Burkina Faso. We identified a number of virus and virus-like sequences from mosquito midgut contents, including 14 coding-complete genome segments and 26 partial sequences. The coding-complete sequences define new viruses in the order Mononegavirales, and the families Flaviviridae, and Totiviridae. The identification of a flavivirus infecting Anopheles mosquitoes broadens our understanding of the evolution and host range of this virus family. This study increases our understanding of virus diversity in general, begins to define the virome of a medically important vector in its natural setting, and lays groundwork for future studies examining the potential impact of these viruses on anopheles biology and disease transmission

Targeted prevention of common mental health disorders in university students: randomised controlled trial of a transdiagnostic trait-focused web-based intervention

Background: A large proportion of university students show symptoms of common mental disorders, such as depression, anxiety, substance use disorders and eating disorders. Novel interventions are required that target underlying factors of multiple disorders.&lt;p&gt;&lt;/p&gt; Aims: To evaluate the efficacy of a transdiagnostic trait-focused web-based intervention aimed at reducing symptoms of common mental disorders in university students.&lt;p&gt;&lt;/p&gt; Method: Students were recruited online (n = 1047, age: M = 21.8, SD = 4.2) and categorised into being at high or low risk for mental disorders based on their personality traits. Participants were allocated to a cognitive-behavioural trait-focused (n = 519) or a control intervention (n = 528) using computerised simple randomisation. Both interventions were fully automated and delivered online (trial registration: ISRCTN14342225). Participants were blinded and outcomes were self-assessed at baseline, at 6 weeks and at 12 weeks after registration. Primary outcomes were current depression and anxiety, assessed on the Patient Health Questionnaire (PHQ9) and Generalised Anxiety Disorder Scale (GAD7). Secondary outcome measures focused on alcohol use, disordered eating, and other outcomes.&lt;p&gt;&lt;/p&gt; Results: Students at high risk were successfully identified using personality indicators and reported poorer mental health. A total of 520 students completed the 6-week follow-up and 401 students completed the 12-week follow-up. Attrition was high across intervention groups, but comparable to other web-based interventions. Mixed effects analyses revealed that at 12-week follow up the trait-focused intervention reduced depression scores by 3.58 (p&#60;.001, 95%CI [5.19, 1.98]) and anxiety scores by 2.87 (p = .018, 95%CI [1.31, 4.43]) in students at high risk. In high-risk students, between group effect sizes were 0.58 (depression) and 0.42 (anxiety). In addition, self-esteem was improved. No changes were observed regarding the use of alcohol or disordered eating.&lt;p&gt;&lt;/p&gt; Conclusions This study suggests that a transdiagnostic web-based intervention for university students targeting underlying personality risk factors may be a promising way of preventing common mental disorders with a low-intensity intervention

C6/36 Aedes albopictus Cells Have a Dysfunctional Antiviral RNA Interference Response

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26–27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level

RNAi Targeting of West Nile Virus in Mosquito Midguts Promotes Virus Diversification

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification

West Nile Virus Genetic Diversity is Maintained during Transmission by Culex pipiens quinquefasciatus Mosquitoes

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes

Cognitive group therapy for depressive students: The case study

The aims of this study were to assess whether a course of cognitive group therapy could help depressed students and to assess whether assimilation analysis offers a useful way of analysing students' progress through therapy. “Johanna” was a patient in a group that was designed for depressive students who had difficulties with their studies. The assimilation of Johanna's problematic experience progressed as the meetings continued from level one (unpleasant thoughts) to level six (solving the problem). Johanna's problematic experience manifested itself as severe and excessive criticism towards herself and her study performance. As the group meetings progressed, Johanna found a new kind of tolerance that increased her determination and assertiveness regarding the studies. The dialogical structure of Johanna's problematic experience changed: she found hope and she was more assertive after the process. The results indicated that this kind of psycho-educational group therapy was an effective method for treating depression. The assimilation analysis offered a useful way of analysing the therapy process

Highly Sensitive and Specific Detection of Rare Variants in Mixed Viral Populations from Massively Parallel Sequence Data

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (<1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved >97% sensitivity and >97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the ∼10 kb genome, including 603 rare variants (<1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses