Clinical and Economic Impact of COVID-19 on Agricultural Workers, Guatemala1.

We evaluated clinical and socioeconomic burdens of respiratory disease in banana farm workers in Guatemala. We offered all eligible workers enrollment during June 15-December 30, 2020, and annually, then tracked them for influenza-like illnesses (ILI) through self-reporting to study nurses, sentinel surveillance at health posts, and absenteeism. Workers who had ILI submitted nasopharyngeal swab specimens for testing for influenza virus, respiratory syncytial virus, and SARS-CoV-2, then completed surveys at days 0, 7, and 28. Through October 10, 2021, a total of 1,833 workers reported 169 ILIs (12.0 cases/100 person-years), and 43 (25.4%) were laboratory-confirmed infections with SARS-CoV-2 (3.1 cases/100 person-years). Workers who had SARS-CoV-2‒positive ILIs reported more frequent anosmia, dysgeusia, difficulty concentrating, and irritability and worse clinical and well-being severity scores than workers who had test result‒negative ILIs. Workers who had positive results also had greater absenteeism and lost income. These results support prioritization of farm workers in Guatemala for COVID-19 vaccination.

The BioFire® RP2.1 Panel Did Not Identify Concurrent Respiratory Virus Infection in Adults with Variable SARS-CoV-2 Disease Severity and Infection Duration.

SARS-CoV-2 emerged in 2019 and rapidly surged into a global pandemic. The rates of concurrent infection with other respiratory pathogens and the effects of possible coinfections on the severity of COVID-19 cases and the length of viral infection are not well defined. In this retrospective study, nasopharyngeal swab samples collected in Colorado between March 2020 and May 2021 from SARS-CoV-2 PCR-positive individuals were tested for a panel of 21 additional respiratory pathogens, including 17 viral and 4 bacterial pathogens. We detected significant positive correlations between levels of SARS-CoV-2 RNA and infectious virus titers for both cohorts, as well as a positive correlation between viral RNA levels and disease severity scores for one cohort. We hypothesized that severe COVID-19 cases and longer SARS-CoV-2 infections may be associated with concurrent respiratory infections. Only one individual exhibited evidence of a concurrent infection- SARS -CoV-2 and human rhinovirus/enterovirus- leading us to conclude that viral respiratory coinfections were uncommon during this time and thus not responsible for the variations in disease severity and infection duration observed in the two cohorts examined. Mask wearing and other public health measures were imposed in Colorado during the time of collection and likely contributed to low rates of coinfection.

Clinical and Economic Impact of COVID-19 on Plantation Workers: Preliminary Results from the Guatemala Agricultural Workers and Respiratory Illness Impact (AGRI) Study.

We evaluated the clinical and socioeconomic burdens of respiratory disease in a cohort of Guatemalan banana plantation workers. All eligible workers were offered enrollment from June 15-December 30, 2020, and annually, then followed for influenza-like illnesses (ILI) through: 1) self-reporting to study nurses, 2) sentinel surveillance at health posts, and 3) absenteeism. Workers with ILI submitted nasopharyngeal swabs for influenza, RSV, and SARS-CoV-2 testing, then completed surveys at days 0, 7, and 28. Through October 10, 2021, 1,833 workers developed 169 ILIs (12.0/100 person-years) and 43 (25.4%) of these ILIs were laboratory-confirmed SARS-CoV-2 (3.1/100 person-years). Workers with SARS-CoV-2-positive ILI reported more anosmia (p<0.01), dysgeusia (p<0.01), difficulty concentrating (p=0.01), and irritability (p=0.01), and greater clinical and well-being severity scores (Flu-iiQ) than test-negative ILIs; they also had greater absenteeism (p<0.01) and lost income (median US$127.1, p<0.01). These results support the prioritization of Guatemalan farm workers for COVID-19 vaccination.

Early Adoption of Longitudinal Surveillance for SARS-CoV-2 among Staff in Long-Term Care Facilities: Prevalence, Virologic and Sequence Analysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and has become a major global pathogen in an astonishingly short period of time. The emergence of SARS-CoV-2 has been notable due to its impacts on residents in long-term care facilities (LTCFs). LTCF residents tend to possess several risk factors for severe outcomes of SARS-CoV-2 infection, including advanced age and the presence of comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. Few studies have focused on the prevalence and transmission dynamics of SARS-CoV-2 among LTCF staff during the early months of the pandemic, prior to mandated surveillance testing. To assess the prevalence and incidence of SARS-CoV-2 among LTCF staff, characterize the extent of asymptomatic infections, and investigate the genomic epidemiology of the virus within these settings, we sampled staff for 8 to 11 weeks at six LTCFs with nasopharyngeal swabs from March through June of 2020. We determined the presence and levels of viral RNA and infectious virus and sequenced 54 nearly complete genomes. Our data revealed that over 50% of infections were asymptomatic/mildly symptomatic and that there was a strongly significant relationship between viral RNA (vRNA) and infectious virus, prolonged infections, and persistent vRNA (4+ weeks) in a subset of individuals, and declining incidence over time. Our data suggest that asymptomatic SARS-CoV-2-infected LTCF staff contributed to virus persistence and transmission within the workplace during the early pandemic period. Genetic epidemiology data generated from samples collected during this period support that SARS-CoV-2 was commonly spread between staff within an LTCF and that multiple-introduction events were less common. IMPORTANCE Our work comprises unique data on the characteristics of SARS-CoV-2 dynamics among staff working at LTCFs in the early months of the SARS-CoV-2 pandemic prior to mandated staff surveillance testing. During this time period, LTCF residents were largely sheltering-in-place. Given that staff were able to leave and return daily and could therefore be a continued source of imported or exported infection, we performed weekly SARS-CoV-2 PCR on nasal swab samples collected from this population. There are limited data from the early months of the pandemic comprising longitudinal surveillance of staff at LTCFs. Our data reveal the surprisingly high level of asymptomatic/presymptomatic infections within this cohort during the early months of the pandemic and show genetic epidemiological analyses that add novel insights into both the origin and transmission of SARS-CoV-2 within LTCFs.

Durable Antibody Responses in Staff at Two Long-Term Care Facilities, during and Post SARS-CoV-2 Outbreaks.

SARS-CoV-2 has had a disproportionate impact on nonhospital health care settings, such as long-term-care facilities (LTCFs). The communal nature of these facilities, paired with the high-risk profile of residents, has resulted in thousands of infections and deaths and a high case fatality rate. To detect presymptomatic infections and identify infected workers, we performed weekly surveillance testing of staff at two LTCFs, which revealed a large outbreak at one of the sites. We collected serum from staff members throughout the study and evaluated it for binding and neutralization to measure seroprevalence, seroconversion, and type and functionality of antibodies. At the site with very few incident infections, we detected that over 40% of the staff had preexisting SARS-CoV-2 neutralizing antibodies, suggesting prior exposure. At the outbreak site, we saw rapid seroconversion following infection. Neutralizing antibody levels were stable for many weeks following infection, suggesting a durable, long-lived response. Receptor-binding domain antibodies and neutralizing antibodies were strongly correlated. The site with high seroprevalence among staff had two unique introductions of SARS-CoV-2 into the facility through seronegative infected staff during the period of study, but these did not result in workplace spread or outbreaks. Together, our results suggest that a high seroprevalence rate among staff can contribute to immunity within a workplace and protect against subsequent infection and spread within a facility. IMPORTANCE Long-term care facilities (LTCFs) have been disproportionately impacted by COVID-19 due to their communal nature and high-risk profile of residents. LTCF staff have the ability to introduce SARS-CoV-2 into the facility, where it can spread, causing outbreaks. We tested staff weekly at two LTCFs and collected blood throughout the study to measure SARS-CoV-2 antibodies. One site had a large outbreak and infected individuals rapidly generated antibodies after infection. At the other site, almost half the staff already had antibodies, suggesting prior infection. The majority of these antibodies bind to the receptor-binding domain of the SARS-CoV-2 spike protein and are potently neutralizing and stable for many months. The non-outbreak site had two unique introductions of SARS-CoV-2 into the facility, but these did not result in workplace spread or outbreaks. Our results reveal that high seroprevalence among staff can contribute to immunity and protect against subsequent infection and spread within a facility.

Association Between COVID-19 Exposure and Self-reported Compliance With Public Health Guidelines Among Essential Employees at an Institution of Higher Education in the US.

Importance: Detailed analysis of infection rates paired with behavioral and employee-reported risk factors is vital to understanding how transmission of SARS-CoV-2 infection may be exacerbated or mitigated in the workplace. Institutions of higher education are heterogeneous work units that supported continued in-person employment during the COVID-19 pandemic, providing a test site for occupational health evaluation. Objective: To evaluate the association between self-reported protective behaviors and prevalence of SARS-CoV-2 infection among essential in-person employees during the first 6 months of the COVID-19 pandemic in the US. Design, Setting, and Participants: This cross-sectional study was conducted from July 13 to September 2, 2020, at an institution of higher education in Fort Collins, Colorado. Employees 18 years or older without symptoms of COVID-19 who identified as essential in-person workers during the first 6 months of the pandemic were included. Participants completed a survey, and blood and nasal swab samples were collected to assess active SARS-CoV-2 infection via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and past infection by serologic testing. Exposure: Self-reported practice of protective behaviors against COVID-19 according to public health guidelines provided to employees. Main Outcomes and Measures: Prevalence of current SARS-CoV-2 infection detected by qRT-PCR or previous SARS-CoV-2 infection detected by an IgG SARS-CoV-2 testing platform. The frequency of protective behavior practices and essential workers' concerns regarding contracting COVID-19 and exposing others were measured based on survey responses. Results: Among 508 participants (305 [60.0%] women, 451 [88.8%] non-Hispanic White individuals; mean [SD] age, 41.1 [12.5] years), there were no qRT-PCR positive test results, and only 2 participants (0.4%) had seroreactive IgG antibodies. Handwashing and mask wearing were reported frequently both at work (480 [94.7%] and 496 [97.8%] participants, respectively) and outside work (465 [91.5%] and 481 [94.7%] participants, respectively). Social distancing was reported less frequently at work (403 [79.5%]) than outside work (465 [91.5%]) (P < .001). Participants were more highly motivated to avoid exposures because of concern about spreading the infection to others (419 [83.0%]) than for personal protection (319 [63.2%]) (P < .001). Conclusions and Relevance: In this cross-sectional study of essential workers at an institution of higher education, when employees reported compliance with public health practices both at and outside work, they were able to operate safely in their work environment during the COVID-19 pandemic.

The amphibian peptide Yodha is virucidal for Zika and dengue viruses.

Zika virus (ZIKV) has emerged as a serious health threat in the Americas and the Caribbean. ZIKV is transmitted by the bite of an infected mosquito, sexual contact, and blood transfusion. ZIKV can also be transmitted to the developing fetus in utero, in some cases resulting in spontaneous abortion, fetal brain abnormalities, and microcephaly. In adults, ZIKV infection has been correlated with Guillain-Barre syndrome. Despite the public health threat posed by ZIKV, neither a vaccine nor antiviral drugs for use in humans are currently available. We have identified an amphibian host defense peptide, Yodha, which has potent virucidal activity against ZIKV. It acts directly on the virus and destroys Zika virus particles within 5 min of exposure. The Yodha peptide was effective against the Asian, African, and South American Zika virus strains and has the potential to be developed as an antiviral therapeutic in the fight against Zika virus. The peptide was also effective against all four dengue virus serotypes. Thus, Yodha peptide could potentially be developed as a pan-therapeutic for Zika and dengue viruses.

Longitudinal Surveillance for SARS-CoV-2 Among Staff in Six Colorado Long-Term Care Facilities: Epidemiologic, Virologic and Sequence Analysis.

BACKGROUND: SARS-CoV-2 emerged in 2019 and has become a major global pathogen. Its emergence is notable due to its impacts on individuals residing within long term care facilities (LTCFs) such as rehabilitation centers and nursing homes. LTCF residents tend to possess several risk factors for more severe SARS-CoV-2 outcomes, including advanced age and multiple comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. METHODS: To assess the prevalence and incidence of SARS-CoV-2 among LTCF workers, determine the extent of asymptomatic SARS-CoV-2 infection, and provide information on the genomic epidemiology of the virus within these unique care settings, we collected nasopharyngeal swabs from workers for 8-11 weeks at six Colorado LTCFs, determined the presence and level of viral RNA and infectious virus within these samples, and sequenced 54 nearly complete genomes. FINDINGS: Our data reveal a strikingly high degree of asymptomatic/mildly symptomatic infection, a strong correlation between viral RNA and infectious virus, prolonged infections and persistent RNA in a subset of individuals, and declining incidence over time. INTERPRETATION: Our data suggest that asymptomatic SARS-CoV-2 infected individuals contribute to virus persistence and transmission within the workplace, due to high levels of virus. Genetic epidemiology revealed that SARS-CoV-2 likely spreads between staff within an LTCF. FUNDING: Colorado State University Colleges of Health and Human Sciences, Veterinary Medicine and Biomedical Sciences, Natural Sciences, and Walter Scott, Jr. College of Engineering, the Columbine Health Systems Center for Healthy Aging, and the National Institute of Allergy and Infectious Diseases.

RNA Helicase LGP2 Negatively Regulates RIG-I Signaling by Preventing TRIM25-Mediated Caspase Activation and Recruitment Domain Ubiquitination.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a family of cytosolic pattern recognition receptors that play a critical role in binding viral RNA and triggering antiviral immune responses. The RLR LGP2 (or DHX58) is a known regulator of the RIG-I signaling pathway; however, the underlying mechanism by which LGP2 regulates RIG-I signaling is poorly understood. To better understand the effects of LGP2 on RIG-I-specific signaling and myeloid cell responses, we probed RIG-I signaling using a highly specific RIG-I agonist to compare transcriptional profiles between WT and Dhx58-/- C57BL\6 bone marrow-derived dendritic cells. Dhx58-/- cells exhibited a marked increase in the magnitude and kinetics of type I interferon (IFN) induction and a broader antiviral response as early as 1 h post-treatment. We determined that LGP2 inhibited RIG-I-mediated IFN-ß, IRF-3, and NF-κB promoter activities, indicating a function upstream of the RLR adaptor protein mitochondrial antiviral signaling. Mutational analysis of LGP2 revealed that RNA binding, ATP hydrolysis, and the C-terminal domain fragment were dispensable for inhibiting RIG-I signaling. Using mass spectrometry, we discovered that LGP2 interacted with the E3 ubiquitin ligase TRIM25. Finally, we determined that LGP2 inhibited the TRIM25-mediated K63-specific ubiquitination of the RIG-I N-terminus required for signaling activation.

Cross-Reactive Dengue Virus Antibodies Augment Zika Virus Infection of Human Placental Macrophages.

Zika virus (ZIKV), which emerged in regions endemic to dengue virus (DENV), is vertically transmitted and results in adverse pregnancy outcomes. Antibodies to DENV can cross-react with ZIKV, but whether these antibodies influence ZIKV vertical transmission remains unclear. Here, we find that DENV antibodies increase ZIKV infection of placental macrophages (Hofbauer cells [HCs]) from 10% to over 80% and enhance infection of human placental explants. ZIKV-anti-DENV antibody complexes increase viral binding and entry into HCs but also result in blunted type I interferon, pro-inflammatory cytokine, and antiviral responses. Additionally, ZIKV infection of HCs and human placental explants is enhanced in an immunoglobulin G subclass-dependent manner, and targeting FcRn reduces ZIKV replication in human placental explants. Collectively, these findings support a role for pre-existing DENV antibodies in enhancement of ZIKV infection of select placental cell types and indicate that pre-existing immunity to DENV should be considered when addressing ZIKV vertical transmission.

Negative regulators of the RIG-I-like receptor signaling pathway.

Upon recognition of specific molecular patterns on microbes, host cells trigger an innate immune response, which culminates in the production of type I interferons, proinflammatory cytokines and chemokines, and restricts pathogen replication and spread within the host. At each stage of this response, there are stimulatory and inhibitory signals that regulate the magnitude, quality, and character of the response. Positive regulation promotes an antiviral state to control and eventually clear infection, whereas negative regulation dampens inflammation and prevents immune-mediated tissue damage. An overexuberant innate response can lead to cell and tissue destruction, and the development of spontaneous autoimmunity. The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I and melanoma differentiation-associated gene 5 (MDA5), belong to a family of cytosolic host RNA helicases that recognize distinct nonself RNA signatures and trigger innate immune responses against several RNA viruses by signaling through the essential adaptor protein mitochondrial antiviral signaling (MAVS). The RLR signaling pathway is tightly regulated to maximize antiviral immunity and minimize immune-mediated pathology. This review highlights contemporary findings on negative regulators of the RLR signaling pathway, with specific focus on the proteins and biological processes that directly regulate RIG-I, MDA5 and MAVS signaling function.

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target.

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations.

Zika Virus Infects Human Placental Macrophages.

The recent Zika virus (ZIKV) outbreak in Brazil has been directly linked to increased cases of microcephaly in newborns. Current evidence indicates that ZIKV is transmitted vertically from mother to fetus. However, the mechanism of intrauterine transmission and the cell types involved remain unknown. We demonstrate that the contemporary ZIKV strain PRVABC59 (PR 2015) infects and replicates in primary human placental macrophages, called Hofbauer cells, and to a lesser extent in cytotrophoblasts, isolated from villous tissue of full-term placentae. Viral replication coincides with induction of type I interferon (IFN), pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. Our results suggest a mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting placental cells and disrupting the placental barrier.

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation.

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.

The receptor tyrosine kinase EphB2 promotes hepatic fibrosis in mice.

UNLABELLED: Beyond the well-defined role of the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinases in developmental processes, cell motility, cell trafficking/adhesion, and cancer, nothing is known about their involvement in liver pathologies. During blood-stage rodent malaria infection we have found that EphB2 transcripts and proteins were up-regulated in the liver, a result likely driven by elevated surface expression on immune cells including macrophages. This was significant for malaria pathogenesis because EphB2(-/-) mice were protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate control mice. This protection was correlated with a defect in the inflammatory potential of hepatocytes from EphB2(-/-) mice resulting in a reduction in adhesion molecules, chemokine/chemokine receptor RNA levels, and infiltration of leukocytes including macrophages/Kupffer cells, which mediate liver fibrosis during rodent malaria infections. These observations are recapitulated in the well-established carbon tetrachloride model of liver fibrosis in which EphB2(-/-) carbon tetrachloride-treated mice showed a significant reduction of liver fibrosis compared to carbon tetrachloride-treated littermate mice. Depletion of macrophages by clodronate-liposomes abrogates liver EphB2 messenger RNA and protein up-regulation and fibrosis in malaria-infected mice. CONCLUSION: During rodent malaria, EphB2 expression promotes malaria-associated liver fibrosis; to our knowledge, our data are the first to implicate the EphB family of receptor tyrosine kinases in liver fibrosis or in the pathogenesis of malaria infection.

Increased Akt signaling in the mosquito fat body increases adult survivorship.

Akt signaling regulates diverse physiologies in a wide range of organisms. We examine the impact of increased Akt signaling in the fat body of 2 mosquito species, the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Overexpression of a myristoylated and active form of A. stephensi and Ae. aegypti Akt in the fat body of transgenic mosquitoes led to activation of the downstream signaling molecules forkhead box O (FOXO) and p70 S6 kinase in a tissue and blood meal-specific manner. In both species, increased Akt signaling in the fat body after blood feeding significantly increased adult survivorship relative to nontransgenic sibling controls. In A. stephensi, survivorship was increased by 15% to 45%, while in Ae. aegypti, it increased 14% to 47%. Transgenic mosquitoes fed only sugar, and thus not expressing active Akt, had no significant difference in survivorship relative to nontransgenic siblings. Expression of active Akt also increased expression of fat body vitellogenin, but the number of viable eggs did not differ significantly between transgenic and nontransgenic controls. This work demonstrates a novel mechanism of enhanced survivorship through increased Akt signaling in the fat bodies of multiple mosquito genera and provides new tools to unlock the molecular underpinnings of aging in eukaryotic organisms.

The innate immune playbook for restricting West Nile virus infection.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes annual epidemics of encephalitic disease throughout the world. Despite the ongoing risk to public health, no approved vaccines or therapies exist for use in humans to prevent or combat WNV infection. The innate immune response is critical for controlling WNV replication, limiting virus-induced pathology, and programming protective humoral and cell-mediated immunity to WNV infection. The RIG-I like receptors, Toll-like receptors, and Nod-like receptors detect and respond to WNV by inducing a potent antiviral defense program, characterized by production of type I IFN, IL-1ß and expression of antiviral effector genes. Recent research efforts have focused on uncovering the mechanisms of innate immune sensing, antiviral effector genes that inhibit WNV, and countermeasures employed by WNV to antagonize innate immune cellular defenses. In this review, we highlight the major research findings pertaining to innate immune regulation of WNV infection.

Manipulating insulin signaling to enhance mosquito reproduction.

BACKGROUND: In the mosquito Aedes aegypti the insulin/insulin growth factor I signaling (IIS) cascade is a key regulator of many physiological processes, including reproduction. Two important reproductive events, steroidogenesis in the ovary and yolk synthesis in the fat body, are regulated by the IIS cascade in mosquitoes. The signaling molecule phosphatase and tensin homolog (PTEN) is a key inhibitor of the IIS cascade that helps modulate the activity of the IIS cascade. In Ae. aegypti, six unique splice variants of AaegPTEN were previously identified, but the role of these splice variants, particularly AaegPTEN3 and 6, were unknown. RESULTS: Knockdown of AaegPTEN or its specific splice variant AaegPTEN6 (the splice variant thought to regulate reproduction in the ovary and fat body) using RNAi led to a 15-63% increase in egg production with no adverse effects on egg viability during the first reproductive cycle. Knockdown of AaegPTEN3, expressed predominantly in the head, had no effect on reproduction. We also characterized the protein expression patterns of these two splice variants during development and in various tissues during a reproductive cycle. CONCLUSION: Previous studies in a range of organisms, including Drosophila melanogaster and Caenorhabditis elegans, have demonstrated that disruption of the IIS cascade leads to decreased reproduction or sterility. In this study we demonstrate that knockdown of the IIS inhibitor PTEN can actually increase reproduction in the mosquito, at least during the first reproductive cycle.