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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Aedes aegypti is a major vector of Zika, dengue, and other arboviruses. Permethrin adulticidal spraying, which targets the voltage-gated sodium channel (VGSC), is commonly done to reduce local mosquito populations and protect humans from exposure to arbovirus pathogens transmitted by this dangerous pest. Permethrin resistance, however, is a growing problem and understanding its underlying molecular basis may identify avenues to combat it. We identified a single G:C polymorphism in pre-miR-33 that was genetically associated with permethrin resistance; resulting isoforms had structural differences that may affect DICER-1/pre-miRNA processing rates. We then assessed the effects of overexpression of pre-miR-33 isoforms on permethrin toxicological phenotypes, VGSC transcript abundance and protein levels for two genetically related mosquito strains. One strain had its naturally high permethrin resistance levels maintained by periodic treatment, and the other was released from selection. VGSC protein levels were lower in the permethrin resistant strain than in the related permethrin-susceptible strain. Overexpression of the G-pre-miR-33 isoform reduced VGSC expression levels in both strains. To further elucidate changes in gene expression associated with permethrin resistance, exome-capture gDNA deep sequencing, genetic association mapping and subsequent gene set enrichment analysis revealed that transport genes, in particular, were selected in resistant versus susceptible mosquitoes. Collectively, these data indicate that miR-33 regulates VGSC expression as part of a nuanced system of neuronal regulation that contributes to a network of heritable features determining permethrin resistance.

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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.

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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.

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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.

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Major histocompatibility complex class II (MHC-II) molecules of multiple species function as cell-entry receptors for the haemagglutinin-like H18 protein of the bat H18N11 influenza A virus, enabling tropism of the viruses in a potentially broad range of vertebrates. However, the function of the neuraminidase-like N11 protein is unknown because it is dispensable for viral infection or the release of H18-pseudotyped viruses. Here, we show that infection of mammalian cells with wild-type H18N11 leads to the emergence of mutant viruses that lack the N11 ectodomain and acquired mutations in H18. An infectious clone of one such mutant virus, designated rP11, appeared to be genetically stable in mice and replicated to higher titres in mice and cell culture compared with wild-type H18N11. In ferrets, rP11 antigen and RNA were detected at low levels in various tissues, including the tonsils, whereas the wild-type virus was not. In Neotropical Jamaican fruit bats, wild-type H18N11 was found in intestinal Peyer's patches and was shed to high concentrations in rectal samples, resulting in viral transmission to naive contact bats. Notably, rP11 also replicated efficiently in bats; however, only restored full-length N11 viruses were transmissible. Our findings suggest that wild-type H18N11 replicates poorly in mice and ferrets and that N11 is a determinant for viral transmission in bats.

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The yellow fever mosquito, Aedes aegypti, serves as the primary vector for epidemic transmission of yellow fever, dengue, Zika (ZIKV), and chikungunya viruses to humans. Control of Ae. aegypti is currently limited to insecticide applications and larval habitat management; however, to combat growing challenges with insecticide resistance, novel genetic approaches for vector population reduction or transmission interruption are being aggressively pursued. The objectives of this study were to assess the ability of the Ae. aegypti antiviral exogenous-small interfering RNA (exo-siRNA) response to inhibit ZIKV infection and transmission, and to identify the optimal RNA interference (RNAi) target region in the ZIKV genome. We accomplished these objectives by in vitro transcription of five long double-stranded RNAs (dsRNAs) from the genome region spanning the NS2B-NS3-NS4A genes, which were the most highly conserved among ZIKV RNA sequences representing both East and West African and Asian-American clades, and evaluation of the ability of these dsRNAs to trigger an effective antiviral exo-siRNA response after intrathoracic injection into Ae. aegypti. In a pilot study, five ZIKV dsRNAs were tested by intrathoracic inoculation of 250 ng dsRNA into groups of approximately 5-day-old mosquitoes. Three days post-inoculation, mosquitoes were provided an infectious blood-meal containing ZIKV strain PRVABC59 (Puerto Rico), MR766 (Uganda), or 41525 (Senegal). On days 7 and 14 post-infection individual whole mosquito bodies were assessed for ZIKV infectious titer by plaque assays. Based on the results of this initial assessment, three dsRNAs were selected for further evaluation of viral loads of matched body and saliva expectorants using a standardized infectious dose of 1 × 107 PFU/mL of each ZIKV strain. Fourteen days post-exposure to ZIKV, paired saliva and carcass samples were harvested from individual mosquitoes and assessed for ZIKV RNA load by qRT-PCR. Injection of each of the three dsRNAs resulted in significant inhibition of replication of all three strains of ZIKV in mosquito bodies and saliva. This study lays critical groundwork for pursuing ZIKV transmission-blocking strategies that exploit the Ae. aegypti exo-siRNA response for arbovirus suppression in natural populations.

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Syrian hamsters (Mesocricetus auratus) are a pathogenesis model for the Nipah virus (NiV), and we sought to determine if they are also susceptible to the Cedar virus (CedPV). Following intranasal inoculation with CedPV, virus replication occurred in the lungs and spleens of infected hamsters, a neutralizing antibody was produced in some hamsters within 8 days post-challenge, and no conspicuous signs of disease occurred. CedPV replicated to a similar magnitude as NiV-Bangladesh in type I IFN-deficient BHK-21 Syrian hamster fibroblasts but replicated 4 logs lower in type I IFN-competent primary Syrian hamster and human pulmonary endothelial cells, a principal target of henipaviruses. The coinfection of these cells with CedPV and NiV failed to rescue CedPV titers and did not diminish NiV titers, suggesting the replication machinery is virus-specific. Type I IFN response transcripts Ifna7, Ddx58, Stat1, Stat2, Ccl5, Cxcl10, Isg20, Irf7, and Iigp1 were all significantly elevated in CedPV-infected hamster endothelial cells, whereas Ifna7 and Iigp1 expression were significantly repressed during NiV infection. These results are consistent with the hypothesis that CedPV's inability to counter the host type I IFN response may, in part, contribute to its lack of pathogenicity. Because NiV causes a fatal disease in Syrian hamsters with similarities to human disease, this model will provide valuable information about the pathogenic mechanisms of henipaviruses.

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The emergence of Zika virus (ZIKV) in the New World has led to more than 200,000 human infections. Perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with Guillain-Barré syndrome (GBS). ZIKV is transmitted to humans by Aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal Aedes sp. mosquitos and non-human primates. In the 1950s and '60s, several bat species were shown to be naturally and experimentally susceptible to ZIKV with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. Because of ZIKV emergence in the Americas, we sought to determine susceptibility of Jamaican fruit bats (Artibeus jamaicensis), one of the most common bats in the New World. Bats were inoculated with ZIKV PRVABC59 but did not show signs of disease. Bats held to 28 days post-inoculation (PI) had detectable antibody by ELISA and viral RNA was detected by qRT-PCR in the brain, saliva and urine in some of the bats. Immunoreactivity using polyclonal anti-ZIKV antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. Tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular Leydig cells. The virus likely localized to the brain via infection of Iba1+ macrophage/microglial cells. Jamaican fruit bats, therefore, may be a useful animal model for the study of ZIKV infection. This work also raises the possibility that bats may have a role in Zika virus ecology in endemic regions, and that ZIKV may pose a wildlife disease threat to bat populations.

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Association mapping of factors that condition pyrethroid resistance in Aedes aegypti has consistently identified genes in multiple functional groups. Toward better understanding of the mechanisms involved, we examined high throughput sequencing data (HTS) from two Aedes aegypti aegypti collections from Merida, Yucatan, Mexico treated with either permethrin or deltamethrin. Exome capture enrichment for coding regions and the AaegL5 annotation were used to identify genes statistically associated with resistance. The frequencies of single nucleotide polymorphisms (SNPs) were compared between resistant and susceptible mosquito pools using a contingency χ2 analysis. The -log10(χ2 p value) was calculated at each SNP site, with a weighted average determined from all sites in each gene. Genes with -log10(χ2 p value) ≥ 4.0 and present among all 3 treatment groups were subjected to gene set enrichment analysis (GSEA). We found that several functional groups were enriched compared to all coding genes. These categories were transport, signal transduction and metabolism, in order from highest to lowest statistical significance. Strikingly, 21 genes with demonstrated association to synaptic function were identified. In the high association group (n = 1,053 genes), several genes were identified that also genetically or physically interact with the voltage-gated sodium channel (VGSC). These genes were eg., CHARLATAN (CHL), a transcriptional regulator, several ankyrin-domain proteins, PUMILIO (PUM), a translational repressor, and NEDD4 (E3 ubiquitin-protein ligase). There were 13 genes that ranked among the top 10%: these included VGSC; CINGULIN, a predicted neuronal gap junction protein, and the aedine ortholog of NERVY (NVY), a transcriptional regulator. Silencing of CHL and NVY followed by standard permethrin bottle bioassays validated their association with permethrin resistance. Importantly, VGSC levels were also reduced about 50% in chl- or nvy-dsRNA treated mosquitoes. These results are consistent with the contribution of a variety of neuronal pathways to pyrethroid resistance in Ae. aegypti.

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