Culex tarsalis is a superior horizontal and vertical vector of West Nile virus (WNV) compared with Culex salinarius. Culex salinarius transmitted WNV genotype NY99 (CT 2741-99 strain) horizontally to suckling mice at significantly lower rates than Cx. tarsalis on Days 8, 9, 10, and 12 post-infection, and Cx. salinarius transmitted WNV genotype NY99 to offspring at a lower vertical transmission infection rate than Cx. tarsalis. Culex tarsalis transmitted WNV genotypes NY99 and WN02 (CT S0084-08 strain) with equal efficiency. Daily percent horizontal transmission of genotype NY99 by Cx. tarsalis-infected per os and by intra-thoracic infection was not significantly different from daily transmission of genotype WN02 from Days 5–23 and Days 2–9 post-infection, respectively. Our findings do not support the previously published hypothesis that genotype NY99 was replaced in the New World by WN02 because of a shorter extrinsic incubation of WN02.
Aging is accompanied by a progressive decline in immune function. Studies have shown age-related decreases in expression and signaling efficiency of Toll-like receptors (TLRs) in monocytes and dendritic cells and dysregulation of macrophage TLR3. Using a multivariable mixed effect model, we report a highly significant increase in TLR5 induced production of IL-8 from monocytes of older individuals (p<0.0001). Elevated IL-8 is accompanied by increased expression of TLR5, both protein and mRNA, and by increased levels of TLR5 mediated phosphorylation of MAPK p38 and ERK. We noted incomplete activation of NF-κB in response to TLR5 signaling in monocytes of elderly donors, as reflected by the absence of an associated increase in the production of TNF-α. Elevated TLR5 may provide a critical mechanism to enhance immune responsiveness in older individuals.
Aging; Toll-like receptors; monocytes; immunosenescence; IL-8; flagellin
West Nile virus (WNV), from the Flaviviridae family, is a re-emerging zoonotic pathogen of medical importance. In humans, WNV infection may cause life-threatening meningoencephalitis or long-term neurologic sequelae. WNV is transmitted by Culex spp mosquitoes and both the arthropod vector and the mammalian host are equipped with antiviral innate immune mechanisms sharing a common phylogeny. As far as the current evidence is able to demonstrate, mosquitoes primarily rely on RNA interference, Toll, Imd and JAK-STAT signaling pathways for limiting viral infection, while mammals are provided with these and other more complex antiviral mechanisms involving antiviral effectors, inflammatory mediators, and cellular responses triggered by highly specialized pathogen detection mechanisms that often resemble their invertebrate ancestry. This mini-review summarizes our current understanding of how the innate immune systems of the vector and the mammalian host react to WNV infection and shape its pathogenesis.
Obligate intracellular bacteria of the Rickettsiales order have evolved to colonize both arthropod and mammalian hosts, but few details are known about the bacterial adaptations that occur during transmission from blood-feeding arthropods to mammals. Here we apply proteomics and transcriptome sequencing to Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, in Ixodes scapularis tick salivary glands, to detect proteins or genes expressed by the pathogen during transmission feeding by the tick. We detected expression of 139 genes, representing 11% of the open reading frames (ORFs) in the A. phagocytophilum genome. The predominant categories of proteins were ribosomal proteins, cell surface proteins, chaperones, and uncharacterized proteins. There was no evidence of DNA replication enzymes, suggesting that most of the A. phagocytophilum cells were no longer dividing. Instead, protein expression reflected conversion to the extracellular, infectious “dense-core” (DC) form. High expression of a DC-specific marker, APH_1235, further suggested this developmental transition in ticks. We showed that blocking APH_1235 with antibodies reduced A. phagocytophilum infection levels in mammalian cell culture. This work represents a starting point for clarifying essential proteins expressed by A. phagocytophilum during transmission from ticks to mammals and demonstrates that the abundantly expressed, DC-associated APH_1235 protein is important during in vivo infection by A. phagocytophilum.
West Nile virus (WNV) is a member of the family Flaviviridae and is a neurotropic pathogen responsible for severe human disease. Flavivirus-infected cells release virus particles that contain variable numbers of precursor membrane (prM) protein molecules at the viral surface. Consequently, antibodies are produced against the prM protein. These antibodies have been shown to activate the infectious potential of fully immature flavivirus particles in vitro. Here, we provide in vivo proof that prM antibodies render immature WNV infectious. Infection with antibody-opsonized immature WNV particles caused disease and death of mice, and infectious WNV was found in the brains and sera.
Semaphorin 7A (Sema7A) is a membrane-associated/secreted protein that plays an essential role in connecting the vertebrate neuronal and immune systems. However, the role of Sema7A has not been elucidated in viral pathogenesis. In this study, we show that abrogation of Sema7A protects mice from lethal West Nile virus (WNV) infection. Mice lacking Sema7A showed increased survival, reduced viral burden, and less blood–brain barrier permeability upon WNV infection. Increased Sema7A levels were evident in murine tissues, as well as in murine cortical neurons and primary human macrophages upon WNV infection. Treatment with Sema7A Ab blocked WNV infection in both of these cell types. Furthermore, Sema7A positively regulates the production of TGF-β1 and Smad6 to facilitate WNV pathogenesis in mice. Collectively, these data elucidate the role of Sema7A in shared signaling pathways used by the immune and nervous systems during viral pathogenesis that may lead to the development of Sema7A-blocking therapies for WNV and possibly other flaviviral infections.
In mammals, circadian and daily rhythms influence nearly all aspects of physiology, ranging from behavior to gene expression. Functional molecular clocks have been described in the murine spleen and splenic NK cells. The aim of our study was to investigate the existence of molecular clock mechanisms in other immune cells. Therefore, we measured the circadian changes in gene expression of clock genes (Per1, Per2, Bmal1, and Clock) and clock-controlled transcription factors (Rev-erbα and Dbp) in splenic enriched macrophages, dendritic cells, and B cells in both mice entrained to a light-dark cycle and under constant environmental conditions. Our study reveals the existence of functional molecular clock mechanisms in splenic macrophages, dendritic cells, and B cells.
Mouse splenic macrophages; dendritic cells; B cells possess functional circadian molecular clocks
The Th17 cytokine, IL-22, regulates host immune responses to extracellular pathogens. Whether IL-22 plays a role in viral infection, however, is poorly understood. We report here that Il22−/− mice were more resistant to lethal West Nile virus (WNV) encephalitis, but had similar viral loads in the periphery compared to wild type (WT) mice. Viral loads, leukocyte infiltrates, proinflammatory cytokines and apoptotic cells in the central nervous system (CNS) of Il22−/− mice were also strikingly reduced. Further examination showed that Cxcr2, a chemokine receptor that plays a non-redundant role in mediating neutrophil migration, was significantly reduced in Il22−/− compared to WT leukocytes. Expression of Cxcr2 ligands, cxcl1 and cxcl5, was lower in Il22−/− brains than wild type mice. Correspondingly, neutrophil migration from the blood into the brain was attenuated following lethal WNV infection of Il22−/− mice. Our results suggest that IL-22 signaling exacerbates lethal WNV encephalitis likely by promoting WNV neuroinvasion.
Circadian rhythms refer to biologic processes that oscillate with a period of approximately 24 hours. These rhythms are sustained by a molecular clock and provide a temporal matrix that ensures the coordination of homeostatic processes with the periodicity of environmental challenges. We demonstrate the circadian molecular clock controls the expression and function of toll like receptor 9 (TLR9). In a vaccination model using TLR9 ligand as adjuvant, mice immunized at the time of enhanced TLR9 responsiveness presented weeks later with an improved adaptive immune response. In a TLR9-dependent mouse model of sepsis, we found that disease severity was dependent on the timing of sepsis induction, coinciding with the daily changes in TLR9 expression and function. These findings unveil a direct molecular link between the circadian and innate immune systems with important implications for immunoprophylaxis and immunotherapy.
Plasmodium spp. are pathogenic to their vertebrate hosts and also apparently, impose a fitness cost on their insect vectors. We show here, however, that Plasmodium-infected mosquitoes survive starvation significantly better than uninfected mosquitoes. This survival advantage during starvation is associated with higher energy resource storage that infected mosquitoes accumulate during period of Plasmodium oocyst development. Microarray analysis revealed that the metabolism of sated mosquitoes is altered in the presence of rapidly growing oocysts, including the down-regulation of several enzymes involved in carbohydrate catabolism. In addition, enhanced expression of several insulin-like peptides was observed in Plasmodium-infected mosquitoes. Blocking insulin-like signaling pathway resulted in impaired Plasmodium development. We conclude that Plasmodium infection alters metabolic pathways in mosquitoes, epitomized by enhanced insulin-like signaling – thereby conferring a survival advantage to the insects during periods of starvation. Manipulation of this pathway might provide new strategies to influence the ability of mosquitoes to survive and transmit the protozoa that cause malaria.
West Nile virus (WNV), a mosquito-borne, single-stranded RNA flavivirus, causes significant human morbidity and mortality in the older population; thus, we investigated the effects of aging on infection with WNV in dendritic cells (DCs). We infected DCs with WNV in vitro and quantified cytokines and chemokines (type I IFN and CXCL10), pathogen recognition receptors RIG-I, and Toll-like receptors 3 and 7. The production of type I IFN was significantly lower in DCs from older donors, compared with younger donors. Although we observed no significant age-related difference in expression or nuclear translocation of signaling molecules in initial antiviral responses, DCs from older donors have diminished induction of late-phase responses (eg, STAT1, IRF7, and IRF1), suggesting defective regulation of type I IFN. Our results identify deficits in critical regulatory pathways in the antiviral response that may contribute to the enhanced susceptibility to viral infections observed in aging.
The maintenance of Borrelia burgdorferi in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in B. burgdorferi plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of B. burgdorferi, has been lacking.
Herein, we examined the expression of rpoS and key lipoprotein genes regulated by RpoS, including ospC, ospA, and dbpA, throughout the entire tick-mammal infectious cycle of B. burgdorferi. Our data revealed that transcription of rpoS, ospC, and dbpA is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of rpoS and dbpA in B. burgdorferi within mouse tissues following borrelial dissemination. However, dbpA transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the dbpBA operon. Our results also provide further evidence for the downregulation of ospA expression during mammalian infection, and the repression of ospC at later phases of mammalian infection by B. burgdorferi.
Our study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of B. burgdorferi to its mammalian host, and is sustained during mammalian infection.
Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.
Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.
The Lyme disease agent, Borrelia burgdorferi, is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8 that reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade resulting in impaired neutrophil phagocytosis and chemotaxis, and diminished Borrelia lysis. Therefore, P8 was renamed the lectin complement pathway inhibitor (TSLPI). TSLPI-silenced ticks, or ticks exposed to TSLPI-immune mice, were hampered in Borrelia transmission. Moreover, Borrelia acquisition and persistence in tick midguts was impaired in ticks feeding on TSLPI-immunized B. burgdorferi-infected mice. Together, our findings suggest an essential role for the lectin complement cascade in Borrelia eradication and demonstrate how a vector-borne pathogen co-opts a vector protein to facilitate early mammalian infection and vector colonization.
MBL; lectin; ficolin; tick immunity; Borrelia burgdorferi; complement; vaccine
West Nile and dengue viruses are (re)emerging mosquito-borne flaviviruses that cause significant morbidity and mortality in man. The identification of mosquito proteins that associate with flaviviruses may provide novel targets to inhibit infection of the vector or block transmission to humans. Here, a tandem affinity purification (TAP) assay was used to identify 18 mosquito proteins that interact with dengue and West Nile capsid, envelope, NS2A or NS2B proteins. We further analyzed the interaction of mosquito cadherin with dengue and West Nile virus envelope protein using co-immunoprecipitation and immunofluorescence. Blocking the function of select mosquito factors, including actin, myosin, PI3-kinase and myosin light chain kinase, reduced both dengue and West Nile virus infection in mosquito cells. We show that the TAP method may be used in insect cells to accurately identify flaviviral-host protein interactions. Our data also provides several targets for interrupting flavivirus infection in mosquito vectors.
dengue; West Nile; mosquito; arbovirus; flavivirus; TAP assay
Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections.
Antibodies; Borrelia burgdorferi; ELISA; Sylvilagus floridanus
Fucosylated structures participate in a wide range of pathological processes in eukaryotes and prokaryotes. The impact of fucose on microbial pathogenesis, however, has been less appreciated in arthropods of medical relevance. Thus, we used the tick-borne bacterium Anaplasma phagocytophilum – the agent of human granulocytic anaplasmosis to understand these processes. Here we show that A. phagocytophilum uses α1,3-fucose to colonize ticks. We demonstrate that A. phagocytophilum modulates the expression of α1,3-fucosyltransferases and gene silencing significantly reduces colonization of tick cells. Acquisition but not transmission of A. phagocytophilum was affected when α1,3-fucosyltransferases were silenced during tick feeding. Our results uncover a novel mechanism of pathogen colonization in arthropods. Decoding mechanisms of pathogen invasion in ticks might expedite the development of new strategies to interfere with the life cycle of A. phagocytophilum.
Anaplasma phagocytophilum causes human granulocytic anaplasmosis, one of the most common tick-borne diseases in North America. This unusual obligate intracellular pathogen selectively persists within polymorphonuclear leukocytes. In this study, using the yeast surrogate model we identified an A. phagocytophilum virulence protein, AptA (Anaplasma phagocytophilum toxin A), that activates mammalian Erk1/2 mitogen activated protein kinase. This activation is important for A. phagocytophilum survival within human neutrophils. AptA interacts with the intermediate filament protein vimentin, which is essential for A. phagocytophilum-induced Erk1/2 activation and infection. A. phagocytophilum infection reorganizes vimentin around the bacterial inclusion, thereby contributing to intracellular survival. These observations reveal a major role for the bacterial protein, AptA, and the host protein, vimentin, in the activation of Erk1/2 during A. phagocytophilum infection.
Polymorphonuclear leukocytes (PMN) are key in innate immunity but their role in viral pathogenesis is incompletely understood. In infection with West Nile virus (WNV), we found that expression of two PMN-attracting chemokines, Cxcl1 and Cxcl2, was rapidly and dramatically elevated in macrophages. PMN are rapidly recruited to the site of WNV infection in mice and support efficient replication of WNV. Mice depleted of PMN after WNV inoculation developed higher viremia and earlier death compared to the control group, suggesting a protective role for PMN. In contrast, when PMN were depleted prior to infection with WNV, and in mice deficient in Cxcr2, a chemokine receptor gene, viremia was reduced and survival was enhanced or delayed. Collectively, these data suggest that PMN have a biphasic response to WNV infection, serving as a reservoir for replication and dissemination in early infection and later contributing to viral clearance.
Neutrophil; PMN; West Nile virus; chemokine; innate immunity
Polymorphonuclear leukocytes (PMNs) are key in innate immunity, but their role in viral pathogenesis is incompletely understood. In infection due to West Nile virus (WNV), we found that expression of 2 PMNattracting chemokines, Cxcl1 and Cxcl2, was rapidly and dramatically elevated in macrophages. PMNs are rapidly recruited to the site of WNV infection in mice and support efficient replication of WNV. Mice depleted of PMNs after WNV inoculation developed higher viremia and experienced earlier death, compared with the control group, which suggest a protective role for PMNs. In contrast, when PMNs were depleted prior to infection with WNV, and in mice deficient in Cxcr2 (a chemokine receptor gene), viremia was reduced and survival was enhanced. Collectively, these data suggest that PMNs have a biphasic response to WNV infection, serving as a reservoir for replication and dissemination in early infection and later contributing to viral clearance.
West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥5-fold differentially up-regulated (DUR) and 202 genes that were ≥10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.
Dengue (DENV), West Nile (WNV) and Yellow Fever (YFV) viruses are responsible for severe human disease and mortality worldwide. There is no vaccine available for dengue or West Nile virus and no specific antiviral is available for any of these viral infections. These viruses are transmitted to humans through the bite of a mosquito vector. Understanding the effects of viral infection on gene expression in the mosquito is crucial to the development of effective antiviral treatments for mosquitoes and may enable researchers to interrupt the human-insect infection cycle. Here we investigate the alterations in gene expression across the entire Aedes aegypti genome during infection with DENV, YFV and WNV over time. We describe several genes that share a similar expression profile during infection with all three viruses. We also use a WNV mosquito cell, mosquito and mouse model to show that virally downregulated genes are inhibitory to infection when overexpressed and that viral regulation of mosquito genes is tissue-specific. Our results provide an extensive amount of data highlighting viral gene targets in the mosquito during infection. This data may also be used to develop broad-spectrum anti-flaviviral treatments in mosquitoes.
Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.
Mosquitoes transmit pathogens that cause infectious diseases of global importance. Techniques to easily introduce genes into mosquitoes, however, limit investigations of the interaction between microbes and their arthropod vectors. We now show that a cationic liposome significantly enhances delivery and expression of plasmid DNA in Aedes aegypti and Anopheles gambiae mosquitoes. We then introduced the genes for Ae. aegypti thioester-containing proteins (AeTEPs), which are involved in the control of flaviviral infection, into mosquitoes using this technique. In vivo transfection of AeTEP-1 into Ae. aegypti significantly reduced dengue virus infection, suggesting that the approach can further our understanding of pathogen-mosquito interactions.