Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance, recovery, and death.

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site-the voltage-gated sodium channel (VGSC)-and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.

Implementation of Telehealth for Psychiatric Care in VA Emergency Departments and Urgent Care Clinics.

Objective: To conduct a mixed-methods evaluation of an emergency telehealth intervention in unscheduled settings (emergency department [ED] and urgent care clinic [UCC]) within the Veterans Health Administration (VHA). Materials and Methods: We used the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework to conduct a mixed-methods evaluation of a novel telehealth program implemented in the VHA (Hospital System) in March 2020. We compared the 3 months preimplementation (December 1, 2019 through February 29, 2020) with the 3 months postimplementation (April 1, 2020 through June 30, 2020), then followed sustainability through January 31, 2021. Qualitative data were obtained from surveys and semistructured interviews of staff and providers and analyzed with thematic analysis. Results: Patient demographics and dispositions were similar pre- and postimplementation. The telemental health intervention was used in 319 (83%) unscheduled mental health consultations in the postimplementation phase. After implementation, we did not detect adverse trends in length of stay, 7-day revisits, or 30-day mortality. Use remained high with 82% (n = 1,010) of all unscheduled mental health consultations performed by telemental health in the sustainability phase. Staff and clinician interviews identified the following themes in the use of telemental health: (1) enhanced efficiency without compromising quality and safety, (2) initial apprehension, (3) the COVID-19 pandemic, and (4) sustainability after resolution of the COVID-19 pandemic. Conclusions: This mixed-methods evaluation of unscheduled telemental health implementation found that its use was feasible, did not impact the safety and efficacy of mental health consultations, and was highly acceptable and sustainable in unscheduled settings.

Craniofacial Autonomic Dysfunction in Migraine: Implications for Treatment and Prognosis.

BACKGROUND: Craniofacial autonomic signs and symptoms (CASS) are relatively underrecognized in the evaluation of migraine headache. Yet, these features provide insight into diagnostic criterion, therapeutic approaches, and overarching disease burden. EVIDENCE ACQUISITION: This review aims to summarize relevant literature evaluating autonomic dysfunction, with focus on CASS, in migraine through targeted literature searches in PubMed. Full articles of original data published between 1974 and 2019 were identified using MeSH terms with no search limits. RESULTS: Although CASS are typically clinically evaluated by subjective patient report, investigational measures of cranial autonomic function have identified marked distinctions between headache attack and attack-free intervals. The presence of CASS during an attack does not differ based on age, sex, or presence of aura. Unilateral CASS may be predictive of longer, more frequent, and/or severe attacks and often co-occur with sensory dysfunction such as allodynia and photophobia. Although limited research has been performed to evaluate targeted therapeutics for migraine with CASS, triptans and onabotulinumtoxinA may demonstrate greater effects in this group. CONCLUSIONS: Migraine remains a debilitating disorder with significant community-wide impacts, necessitating continued evaluation of contributing features. Consideration of CASS provides important insight into potential treatment approaches and the effectiveness of novel therapeutic interventions aimed at improving overall disease burden. However, further investigation is needed to fully understand primary craniofacial features in migraine, and how these might inform individualized treatment decisions.

Alternative patterns of sex chromosome differentiation in Aedes aegypti (L).

BACKGROUND: Some populations of West African Aedes aegypti, the dengue and zika vector, are reproductively incompatible; our earlier study showed that divergence and rearrangements of genes on chromosome 1, which bears the sex locus (M), may be involved. We also previously described a proposed cryptic subspecies SenAae (PK10, Senegal) that had many more high inter-sex FST genes on chromosome 1 than did Ae.aegypti aegypti (Aaa, Pai Lom, Thailand). The current work more thoroughly explores the significance of those findings. RESULTS: Intersex standardized variance (FST) of single nucleotide polymorphisms (SNPs) was characterized from genomic exome capture libraries of both sexes in representative natural populations of Aaa and SenAae. Our goal was to identify SNPs that varied in frequency between males and females, and most were expected to occur on chromosome 1. Use of the assembled AaegL4 reference alleviated the previous problem of unmapped genes. Because the M locus gene nix was not captured and not present in AaegL4, the male-determining locus, per se, was not explored. Sex-associated genes were those with FST values ≥ 0.100 and/or with increased expected heterozygosity (H exp , one-sided T-test, p < 0.05) in males. There were 85 genes common to both collections with high inter-sex FST values; all genes but one were located on chromosome 1. Aaa showed the expected cluster of high inter-sex FST genes proximal to the M locus, whereas SenAae had inter-sex FST genes along the length of chromosome 1. In addition, the Aaa M-locus proximal region showed increased H exp levels in males, whereas SenAae did not. In SenAae, chromosomal rearrangements and subsequent suppressed recombination may have accelerated X-Y differentiation. CONCLUSIONS: The evidence presented here is consistent with differential evolution of proto-Y chromosomes in Aaa and SenAae.

Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Arterial stiffness, oxidative stress, and smoke exposure in wildland firefighters.

OBJECTIVES: To assess the association between exposure, oxidative stress, symptoms, and cardiorespiratory function in wildland firefighters. METHODS: We studied two Interagency Hotshot Crews with questionnaires, pulse wave analysis for arterial stiffness, spirometry, urinary 8-iso-prostaglandin F2α (8-isoprostane) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), and the smoke exposure marker (urinary levoglucosan). Arterial stiffness was assessed by examining levels of the aortic augmentation index, expressed as a percentage. An oxidative stress score comprising the average of z-scores created for 8-OHdG and 8-isoprostane was calculated. RESULTS: Mean augmentation index % was higher for participants with higher oxidative stress scores after adjusting for smoking status. Specifically for every one unit increase in oxidative stress score the augmentation index % increased 10.5% (95% CI: 2.5, 18.5%). Higher mean lower respiratory symptom score was associated with lower percent predicted forced expiratory volume in one second/forced vital capacity. CONCLUSIONS: Biomarkers of oxidative stress may serve as indicators of arterial stiffness in wildland firefighters.

Discrimination of Jamaican fruit bat lymphocytes by flow cytometry.

Bats are natural reservoir hosts of many important zoonotic viruses but because there are few immunological reagents and breeding colonies available for infectious disease research, little is known about their immune responses to infection. We established a breeding colony Jamaican fruit bats ( Artibeus jamaicensis ) to study bat virology and immunology. The species is used as a natural reservoir model for H18N11 influenza A virus, and as a surrogate model for SARS-CoV-2, MERS-CoV and Tacaribe virus. As part of our ongoing efforts to develop this model organism, we sought to identify commercially available monoclonal antibodies (mAb) for profiling Jamaican fruit bat lymphocytes. We identified several cross-reactive mAb that can be used to identify T and B cells; however, we were unable to identify mAb for three informative T cell markers, CD3γ, CD4 and CD8α. We targeted these markers for the generation of hybridomas, and identified several clones to each that can be used with flow cytometry and fluorescence microscopy. Specificity of the monoclonal antibodies was validated by sorting lymphocytes, followed by PCR identification of confirmatory transcripts. Spleens of Jamaican fruit bats possess about half the number of T cells than do human or mouse spleens, and we identified an unusual population of cells that expressed the B cell marker CD19 and the T cell marker CD3. The availability of these monoclonal antibodies will permit a more thorough examination of adaptive immune responses in Jamaican fruit bats that should help clarify how the bats control viral infections and without disease. Importance: Bats naturally host a number of viruses without disease, but which can cause significant disease in humans. Virtually nothing is known about adaptive immune responses in bats because of a lack of immunological tools to examine such responses. We have begun to address this deficiency by identifying several commercially available monoclonal antibodies to human and mouse antigens that are cross-reactive to Jamaican fruit bat lymphocyte orthologs. We also generated monoclonal antibodies to Jamaican fruit bat CD3γ, CD4 and CD8α that are suitable for identifying T cell subsets by flow cytometry and immunofluorescent staining of fixed tissues. Together, these reagents will allow a more detailed examination of lymphocyte populations in Jamaican fruit bats.

Factors Associated with Acute Telemental Health Consultations in Older Veterans.

Introduction: The United States Veterans Health Administration is a leader in the use of telemental health (TMH) to enhance access to mental healthcare amidst a nationwide shortage of mental health professionals. The Tennessee Valley Veterans Affairs (VA) Health System piloted TMH in its emergency department (ED) and urgent care clinic (UCC) in 2019, with full 24/7 availability beginning March 1, 2020. Following implementation, preliminary data demonstrated that veterans ≥65 years old were less likely to receive TMH than younger patients. We sought to examine factors associated with older veterans receiving TMH consultations in acute, unscheduled, outpatient settings to identify limitations in the current process. Methods: This was a retrospective cohort study conducted within the Tennessee Valley VA Health System. We included veterans ≥55 years who received a mental health consultation in the ED or UCC from April 1, 2020-September 30, 2022. Telemental health was administered by a mental health clinician (attending physician, resident physician, nurse practitioner, or physician assistant) via iPad, whereas in-person evaluations were performed in the ED. We examined the influence of patient demographics, visit timing, chief complaint, and psychiatric history on TMH, using multivariable logistic regression. Results: Of the 254 patients included in this analysis, 177 (69.7%) received TMH. Veterans with high-risk chief complaints (suicidal ideation, homicidal ideation, or agitation) were less likely to receive TMH consultation (adjusted odds ratio [AOR]: 0.47, 95% confidence interval [CI] 0.24-0.95). Compared to attending physicians, nurse practitioners and physician assistants were associated with increased TMH use (AOR 4.81, 95% CI 2.04-11.36), whereas consultation by resident physicians was associated with decreased TMH use (AOR 0.04, 95% CI 0.00-0.59). The UCC used TMH for all but one encounter. Patient characteristics including their visit timing, gender, additional medical complaints, comorbidity burden, and number of psychoactive medications did not influence use of TMH. Conclusion: High-risk chief complaints, location, and type of mental health clinician may be key determinants of telemental health use in older adults. This may help expand mental healthcare access to areas with a shortage of mental health professionals and prevent potentially avoidable transfers in low-acuity situations. Further studies and interventions may optimize TMH for older patients to ensure safe, equitable mental health care.

A single-cell atlas of the Culex tarsalis midgut during West Nile virus infection.

The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and associated virus infection dynamics are scarce, and in Culex tarsalis - an extremely efficient vector of West Nile virus (WNV) - nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprised of 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells and cells enriched for mitochondrial genes, suggesting enhanced replication in these populations. In contrast, proliferating intestinal stem cell (ISC) populations had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in viral control. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2, R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level.

Dishevelled Has Anti-Viral Activity in Rift Valley Fever Virus Infected Aedes aegypti.

Mosquitoes in the genera Aedes and Culex are vectors of Rift Valley fever virus (RVFV), which emerges in periodic epidemics in Africa and Saudi Arabia. Factors that influence the transmission dynamics of RVFV are not well characterized. To address this, we interrogated mosquito host-signaling responses through analysis of differentially expressed genes (DEGs) in two mosquito species with marked differences in RVFV vector competence: Aedes aegypti (Aae, low competence) and Culex tarsalis (Cxt, high competence). Mosquito-host transcripts related to three different signaling pathways were investigated. Selected genes from the Wingless (Wg, WNT-beta-catenin) pathway, which is a conserved regulator of cell proliferation and differentiation, were assessed. One of these, dishevelled (DSH), differentially regulates progression/inhibition of the WNT and JNK (c-Jun N-terminal Kinase) pathways. A negative regulator of the JNK-signaling pathway, puckered, was also assessed. Lastly, Janus kinase/signal transducers and activators of transcription (JAK-STAT) are important for innate immunity; in this context, we tested domeless levels. Here, individual Aae and Cxt were exposed to RVFV MP-12 via oral bloodmeals and held for 14 days. Robust decreases in DEGs in both Aae and Cxt were observed. In particular, Aae DSH expression, but not Cxt DSH, was correlated to the presence/absence of viral RNA at 14 days post-challenge (dpc). Moreover, there was an inverse relationship between the viral copy number and aaeDSH expression. DSH silencing resulted in increased viral copy numbers compared to controls at 3 dpc, consistent with a role for aaeDSH in antiviral immunity. Analysis of cis-regulatory regions for the genes of interest revealed clues to upstream regulation of these pathways.

Telemental health in emergency care settings: A qualitative analysis of considerations for sustainability and spread.

OBJECTIVES: Following rapid uptake of telehealth during the COVID-19 pandemic, we examined barriers and facilitators for sustainability and spread of telemental health video (TMH-V) as policies regarding precautions from the pandemic waned. METHODS: We conducted a qualitative study using semistructured interviews and observations guided by RE-AIM. We asked four groups, local clinicians, facility leadership, Veterans, and external partners, about barriers and facilitators impacting patient willingness to engage in TMH-V (reach), quality of care (effectiveness), barriers and facilitators impacting provider uptake (adoption), possible adaptations to TMH-V (implementation), and possibilities for long-term use of TMH-V (maintenance). Interviews were recorded, transcribed, and analyzed using framework analysis. We also observed TMH-V encounters in one emergency department (ED) and one urgent care (UC) to understand how clinicians and Veterans engaged with the technology. RESULTS: We conducted 35 interviews with ED/UC clinicians and staff (n = 10), clinical and facility leadership (n = 7), Veterans (n = 5), and external partners (n = 13), January-May 2022. We completed 10 observations. All interviewees were satisfied with the TMH-V program, and interviewees highlighted increased comfort discussing difficult topics for Veterans (reach). Clinicians identified that TMH-V allowed for cross-coverage across sites as well as increased safety and flexibility for clinicians (adoption). Opportunities for improvement include alleviating technological burdens for on-site staff, electronic health record (EHR) modifications to accurately capture workload and modality (telehealth vs. in-person), and standardizing protocols to streamline communication between on-site and remote clinical staff (implementation). Finally, interviewees encouraged its spread (maintenance) and thought there was great potential for service expansion. CONCLUSIONS: Interviewees expressed support for continuing TMH-V locally and spread to other sites. Ensuring adequate infrastructure (e.g., EHR integration and technology support) and workforce capacity are key for successful spread. Given the shortage of mental health (MH) clinicians in rural settings, TMH-V represents a promising intervention to increase the access to high-quality emergency MH care.

The effect of telemental versus in-person mental health consults in the emergency department on 30-day utilization and processes of care.

OBJECTIVES: We sought to characterize how telemental health (TMH) versus in-person mental health consults affected 30-day postevaluation utilization outcomes and processes of care in Veterans presenting to the emergency department (ED) and urgent care clinic (UCC) with acute psychiatric complaints. METHODS: This exploratory retrospective cohort study was conducted in an ED and UCC located in a single Veterans Affairs system. A mental health provider administered TMH via iPad. The primary outcome was a composite of return ED/UCC visits, rehospitalizations, or death within 30 days. The following processes of care were collected during the index visit: changes to home psychiatric medications, admission, involuntary psychiatric hold placement, parenteral benzodiazepine or antipsychotic medication use, and physical restraints or seclusion. Data were abstracted from the Veterans Affairs electronic health record and the Clinical Data Warehouse. Multivariable logistic regression was performed. Adjusted odds ratios (aORs) with their 95% confidence intervals (95% CIs) were reported. RESULTS: Of the 496 Veterans in this analysis, 346 (69.8%) received TMH, and 150 (30.2%) received an in-person mental health evaluation. There was no significant difference in the primary outcome of 30-day return ED/UCC, rehospitalization, or death (aOR 1.47, 95% CI 0.87-2.49) between the TMH and in-person groups. TMH was significantly associated with increased ED/UCC length of stay (aOR 1.46, 95% CI 1.03-2.06) and decreased use of involuntary psychiatric holds (aOR 0.42, 95% CI 0.23-0.75). There were no associations between TMH and the other processes-of-care outcomes. CONCLUSIONS: TMH was not significantly associated with the 30-day composite outcome of return ED/UCC visits, rehospitalizations, and death compared with traditional in-person mental health evaluations. TMH was significantly associated with increased ED/UCC length of stay and decreased odds of placing an involuntary psychiatric hold. Future studies are required to confirm these findings and, if confirmed, explore the potential mechanisms for these associations.

Qualitatively Exploring Mental Health Attitude Changes among Emerging Adult Motivational Interviewing after One Motivational Enhanced Interview.

Emerging adult military members and veterans (MMV) are experiencing many transitions (e.g., adulthood, military). The sum of these changes can cause stress, anxiety, and mental health challenges. Stigma of mental health and treatment exists, and military populations are often not seeking or engaging in appropriate care. Recent research emphasizes the need to uncover mental health attitudes and self-stigma barriers regarding help seeking. We evaluated the impact of a single motivational-interviewing enhanced interview with 26 MMV, all who reported high risk substance use. In 75-minute interviews with the primary focus of discussing their experiences regarding mental health, substance use, and identity development, the interviewer incorporated motivational interviewing strategies (e.g., affirmations, complex reflections). Participants shared their developmental experiences, stressors transitioning, and barriers and stigma around mental health treatment. Participants completed a survey which included a variety of standardized measures and open-ended questions two weeks before and after the interview. Qualitative follow-up data via open ended questions shows the session was well received by participants as they could share their stories, think critically about their military experiences, and brainstorm solutions for mental health care. We conclude that using individual, confidential interviews to discuss sensitive topics for data collection with MMV is an area to continue developing. Conducting qualitative research with motivational interviewing strategies has the potential to be twofold: advance scholarship and inform practitioners, but also serve as a therapeutic platform for some participants. Supplementary Information: The online version contains supplementary material available at 10.1007/s10615-022-00837-z.

Adolescent Cannabis Use Among Youth in ZIP Codes with Medical Dispensaries.

Introduction: In the United States, 19 states permit recreational use of cannabis, with 16 more permitting medical use (Marijuana Policy Project, 2021). Concerns remain about whether liberalized policies result in increased adolescent cannabis use. To date, limited evidence exists that the statewide prevalence of adolescent cannabis use increased in states with liberalized policies. However, analyses at local levels show some negative impacts. Thus, we analyzed if living in a ZIP code with a dispensary (ZCWD) was associated with adolescent cannabis use. Methods: Dispensary ZIP codes from public records were matched to self- reported ZIP codes on the Illinois Youth Survey (IYS). We compared past 30-day and past-year cannabis use among youth living in a ZCWD and not living in a ZCWD. Results: About one in eight adolescents (12.8%, n = 1,348) in the weighted sample (n=10,569) resided in a ZCWD. Overall, past 30-day use was lower among youth who lived in ZIP codes with dispensaries (OR = .69, p < .05), with variation by grade. For example, only 10th (OR = .62, p < .05) and 12th graders (OR = .59, p < .05) living in a ZCWD had lower odds of past 30-day cannabis use. Additionally, only 12th graders in a ZCWD had lower odds of past-year use (OR = .70, p < .05). Finally, suburban youth living in a ZCWD also had lower odds of cannabis use (OR = .54, p < .01). Conclusion/Discussion: Cannabis use was significantly lower among 10th and 12th graders living in a ZCWD. Additional research should continue to monitor evolving state policies and whether they are associated with adolescent cannabis use.

Safety study of Rift Valley Fever human vaccine candidate (DDVax) in mosquitoes.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen with significant human and veterinary health consequences that periodically emerges in epizootics. RVFV causes fetal loss and death in ruminants and in humans can lead to liver and renal disease, delayed-onset encephalitis, retinitis, and in some cases severe haemorrhagic fever. A live attenuated vaccine candidate (DDVax), was developed by the deletion of the virulence factors NSs and NSm from a clinical isolate, ZH501, and has proven safe and immunogenic in rodents, pregnant sheep and non-human primates. Deletion of NSm also severely restricted mosquito midgut infection and inhibited vector-borne transmission. To demonstrate environmental safety, this study investigated the replication, dissemination and transmission efficiency of DDVax in mosquitoes following oral exposure compared to RVFV strains MP-12 and ZH501. Infection and dissemination profiles were also measured in mosquitoes 7 days after they fed on goats inoculated with DDvax or MP-12. We hypothesized that DDVax would infect mosquitoes at significantly lower rates than other RVFV strains and, due to lack of NSm, be transmission incompetent. Exposure of Ae. aegypti and Cx. tarsalis to 8 log10 plaque forming units (PFU)/ml DDVax by artificial bloodmeal resulted in significantly reduced DDVax infection rates in mosquito bodies compared to controls. Plaque assays indicated negligible transmission of infectious DDVax in Cx. tarsalis saliva (1/140 sampled) and none in Ae. aegypti saliva (0/120). Serum from goats inoculated with DDVax or MP-12 did not harbour detectable infectious virus by plaque assay at 1, 2 or 3 days post-inoculation. Infectious virus was, however, recovered from Aedes and Culex bodies that fed on goats vaccinated with MP-12 (13.8% and 4.6%, respectively), but strikingly, DDvax-positive mosquito bodies were greatly reduced (4%, and 0%, respectively). Furthermore, DDVax did not disseminate to legs/wings in any of the goat-fed mosquitoes. Collectively, these results are consistent with a beneficial environmental safety profile.

RNA-seq analyses of blood-induced changes in gene expression in the mosquito vector species, Aedes aegypti.

BACKGROUND: Hematophagy is a common trait of insect vectors of disease. Extensive genome-wide transcriptional changes occur in mosquitoes after blood meals, and these are related to digestive and reproductive processes, among others. Studies of these changes are expected to reveal molecular targets for novel vector control and pathogen transmission-blocking strategies. The mosquito Aedes aegypti (Diptera, Culicidae), a vector of Dengue viruses, Yellow Fever Virus (YFV) and Chikungunya virus (CV), is the subject of this study to look at genome-wide changes in gene expression following a blood meal. RESULTS: Transcriptional changes that follow a blood meal in Ae. aegypti females were explored using RNA-seq technology. Over 30% of more than 18,000 investigated transcripts accumulate differentially in mosquitoes at five hours after a blood meal when compared to those fed only on sugar. Forty transcripts accumulate only in blood-fed mosquitoes. The list of regulated transcripts correlates with an enhancement of digestive activity and a suppression of environmental stimuli perception and innate immunity. The alignment of more than 65 million high-quality short reads to the Ae. aegypti reference genome permitted the refinement of the current annotation of transcript boundaries, as well as the discovery of novel transcripts, exons and splicing variants. Cis-regulatory elements (CRE) and cis-regulatory modules (CRM) enriched significantly at the 5'end flanking sequences of blood meal-regulated genes were identified. CONCLUSIONS: This study provides the first global view of the changes in transcript accumulation elicited by a blood meal in Ae. aegypti females. This information permitted the identification of classes of potentially co-regulated genes and a description of biochemical and physiological events that occur immediately after blood feeding. The data presented here serve as a basis for novel vector control and pathogen transmission-blocking strategies including those in which the vectors are modified genetically to express anti-pathogen effector molecules.

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

BACKGROUND: 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 Ae. aegypti, we deep-sequenced small non-coding RNAs. Triplicate biological replicates were used so that rigorous statistical metrics could be applied. RESULTS: 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. CONCLUSIONS: 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 Ae. aegypti.

From Global to Local-New Insights into Features of Pyrethroid Detoxification in Vector Mosquitoes.

The threat of mosquito-borne diseases continues to be a problem for public health in subtropical and tropical regions of the world; in response, there has been increased use of adulticidal insecticides, such as pyrethroids, in human habitation areas over the last thirty years. As a result, the prevalence of pyrethroid-resistant genetic markers in natural mosquito populations has increased at an alarming rate. This review details recent advances in the understanding of specific mechanisms associated with pyrethroid resistance, with emphasis on features of insecticide detoxification and the interdependence of multiple cellular pathways. Together, these advances add important context to the understanding of the processes that are selected in resistant mosquitoes. Specifically, before pyrethroids bind to their targets on motoneurons, they must first permeate the outer cuticle and diffuse to inner tissues. Resistant mosquitoes have evolved detoxification mechanisms that rely on cytochrome P450s (CYP), esterases, carboxyesterases, and other oxidation/reduction (redox) components to effectively detoxify pyrethroids to nontoxic breakdown products that are then excreted. Enhanced resistance mechanisms have evolved to include alteration of gene copy number, transcriptional and post-transcriptional regulation of gene expression, as well as changes to cellular signaling mechanisms. Here, we outline the variety of ways in which detoxification has been selected in various mosquito populations, as well as key gene categories involved. Pathways associated with potential new genes of interest are proposed. Consideration of multiple cellular pathways could provide opportunities for development of new insecticides.

Opioid use at the transition to emerging adulthood: A latent class analysis of non-medical use of prescription opioids and heroin use.

BACKGROUND: Although rates of nonmedical opioid use are highest in late adolescence and emerging adulthood, efforts to understand the extent of the heterogeneity in opioid misuse during this time have been limited. The current study aimed to derive and define typologies of opioid use in high school students at the onset of emerging adulthood. METHODS: Survey responses from a statewide sample of high school students aged 18 and 19 (N = 26,223) were analyzed. Group-based comparisons between participants reporting opioid use and those not reporting opioid use were conducted. Among those reporting opioid use (n = 1,636), we conducted a latent class analysis (LCA) to identify heterogeneous subgroups of opioid users on the basis of non-medical use of prescription opioids (NMUPO) and heroin use. The resulting classes were then compared across various risk and protective factors using multinominal logistic regression. RESULTS: Consistent differences were observed between participants using opioids and participants not using opioids, with moderate to large effect sizes. Results from LCA revealed three subclasses: NMUPO-Any Use, NMUPO To Get High, and Heroin Use. Subclass differences were observed for non-opioid substance use, mental health, and demographics. CONCLUSIONS: Findings from this study underscore the variability of youth who engage in opioid use in late adolescence. Results also indicate that opioid use during adolescence is likely indicative of a broader set of substance use and mental health issues.