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.

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.

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.

Background

Dengue virus (DENV) and West Nile virus (WNV), close siblings of the Flaviviridae family, are the causative agents of Dengue hemorraghic shock or West Nile meningoencephalitis respectively. Vaccines against these two flaviviruses are currently unavailable. Interferon- Stimulated Gene 15 (ISG15), encoding an ubiquitin-like protein, is significantly induced by type I interferons or viral infections. Its roles in viral infections, however, vary with viruses, being either anti- or pro-viral. The exact roles of ISG15 in DENV and WNV infections remain unknown. In the current study, we evaluated the relevancies of ISG15 to DENV and WNV infection of a mouse macrophage cell line RAW264.7.

Findings

Quantitative PCR showed that mouse Isg15 was dramatically induced in DENV or WNV- infected RAW264.7 cells compared with non-infected cells. Isg15 and two other Jak-Stat related genes, Socs1 and Socs3, were silenced using siRNA mediated RNA interference. The intracellular DENV and WNV loads, as determined by quantitative PCR, were significantly higher in Isg15 silenced cells than control cells. The expression levels of interferon beta 1 (Ifnb1) were increased significantly in Isg15, Socs1 or Socs3 siRNA treated cells. Further investigation indicated that protein modification by ISG15, so called ISGylation, was significantly enhanced in DENV-infected cells compared to that in non-infected cells.

Conclusions

These findings suggest that ISG15 plays an anti-DENV/WNV function via protein ISGylation.

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.

Author Summary

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.

SUMMARY

West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homologue of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells, and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF) from Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi) significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens.

Author Summary

Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Safe and effective vaccines against most tick-borne pathogens are not currently available. Typical vaccines target microbes directly, using extracts of the organism, or recombinant antigens as the immunogen; the transmission of tick-borne pathogens can also theoretically be prevented by interfering with the ability of ticks to feed on a mammalian host. In this study, we have characterized a putative histamine release factor (tHRF) from I. scapularis ticks, the predominant vector of B. burgdorferi, the agent of Lyme disease in North America. Our results suggested that tHRF is presented in tick saliva and critical for tick feeding; blocking tHRF markedly reduced the efficiency of tick feeding, and reduced the B. burgdorferi burden in mice. This finding provides novel insights into the molecular mechanisms of tick feeding and provides a potential vaccine target to block tick feeding and pathogen transmission.

Traditionally, vaccines directly target a pathogen or microbial toxin. Lyme disease, caused by Borrelia burgdorferi, is a tick-borne illness for which a human vaccine is not currently available. B. burgdorferi binds a tick salivary protein, Salp15, during transmission from the vector, and this interaction facilitates infection of mice. We now show that Salp15-antiserum significantly protected mice from B. burgdorferi infection. Salp15-antiserum also markedly enhanced the protective capacity of antibodies against B. burgdorferi antigens, such as OspA or OspC. Mice actively immunized with Salp15 were also significantly protected from tick-borne Borrelia. In vitro assays showed that Salp15-antiserum increased the clearance of Salp15-coated B. burgdorferi by phagocytes, suggesting a mechanism of action. Vaccination with a vector molecule that a microbe requires for infection of the mammalian host suggests a new strategy for the prevention of Lyme disease, and this paradigm may be applicable to numerous arthropod-borne pathogens of medical importance.

West Nile virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Our data show that interleukin-10 (IL-10) is dramatically elevated both in vitro and in vivo following WNV infection. Consistent with an etiologic role of IL-10 in WNV pathogenesis, we find that WNV infection is markedly diminished in IL-10 deficient (IL-10−/−) mice, and pharmacologic blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice. Increased production of antiviral cytokines in IL-10−/− mice is associated with more efficient control of WNV infection. Moreover, CD4+ T cells produce copious amounts of IL-10, and may be an important cellular source of IL-10 during WNV infection in vivo. In conclusion, IL-10 signaling plays a negative role in immunity against WNV infection, and blockade of IL-10 signaling by genetic or pharmacologic means helps to control viral infection, suggesting a novel anti-WNV therapeutic strategy.

Author Summary

West Nile virus (WNV), a mosquito-transmitted RNA virus, is a worldwide cause of severe human and animal infection. Mammalian host immune responses to WNV infection are not completely understood and a vaccine or specific therapy is unavailable for use in humans. In the present study, we investigated the putative regulatory role of interleukin-10 (IL-10) during WNV infection in mice. We found that IL-10 signaling facilitates WNV infection and suppresses antiviral cytokine production in response to viral infection. Interestingly, blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice, suggesting a potentially novel therapeutic strategy to combat WNV infection. In addition, we found that CD4+ T cells produce a significant amount of IL-10 during WNV infection, providing a more accurate cellular target for IL-10 signaling inhibition. IL-10 also plays a critical role in suppression of excessive inflammation and immunopathology caused by autoimmune diseases or host immune system responses to infections; therefore, safety and efficacy of IL-10 signaling blockade as a therapeutic strategy against WNV infection deserves consideration.

Borrelia burgdorferi invasion of mammalian joints results in genesis of Lyme arthritis. Other than spirochete lipids, existence of protein antigens, which are abundant in joints and participate in B. burgdorferi-induced host inflammatory response, is unknown. Here, we report that major products of the B. burgdorferi basic membrane protein (bmp) A/B operon that are induced in murine and human joints, possess inflammatory properties. Compared to the wild type B. burgdorferi, an isogenic bmpA/B mutant induced significantly lower levels of pro-inflammatory cytokines TNF-α and IL-1β in cultured human synovial cells, which could be restored using bmpA/B-complemented mutants, and more directly, upon addition of recombinant BmpA, but not BmpB or control spirochete proteins. Non-lipidated and lipidated versions of BmpA induced similar levels of cytokines, and remained unaffected by treatment with lipopolysaccharide inhibitor, polymyxin B. The bmpA/B mutant was also impaired in the induction of NF-κB and p38 MAP kinase signaling pathways in synovial cells, which were activated by non-lipidated BmpA. These results show that a protein moiety of BmpA can induce cytokine responses in synovial cells via activation of the NF-κB and p38 MAP kinase pathways and thus, could potentially contribute to the genesis of Lyme arthritis.

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

Author Summary

Lyme borreliosis is caused by the spirochete Borrelia burgdorferi and is transmitted through ticks. Since its discovery approximately 30 years ago it has become the most important vector-borne disease in the Western world. The pathogenesis of this complex zoonosis is still not entirely understood. We here demonstrate that the urokinase receptor (uPAR) is upregulated in mice and humans upon exposure to B. burgdorferi in vitro and in vivo. Importantly, we describe the function of uPAR in the immune response against the spirochete; using uPAR knock-out mice, we show that uPAR plays an important role in phagocytosis of B. burgdorferi by leukocytes both in vitro as well as in vivo. In addition, we show that the mechanism by which uPAR is involved in the phagocytosis of B. burgdorferi is independent of ligation to its natural ligand uPA or uPAR's role in fibrinolysis. Our study contributes to the understanding of the pathogenesis of Lyme borreliosis and might contribute to the development of innovative novel treatment strategies for Lyme borreliosis.

West Nile virus (WNV) is the most-common cause of mosquito-borne encephalitis in the United States. Invasion of the brain by WNV is influenced by viral and host factors, and the molecular mechanism underlying disruption of the blood-brain barrier is likely multifactorial. Here we show that matrix metalloproteinase 9 (MMP9) is involved in WNV entry into the brain by enhancing blood-brain barrier permeability. Murine MMP9 expression was induced in the circulation shortly after WNV infection, and the protein levels remained high even when viremia subsided. In the murine brain, MMP9 expression and its enzymatic activity were upregulated and MMP9 was shown to partly localize to the blood vessels. Interestingly, we also found that cerebrospinal fluid from patients suffering from WNV contained increased MMP9 levels. The peripheral viremia and expression of host cytokines were not altered in MMP9−/− mice; however, these animals were protected from lethal WNV challenge. The resistance of MMP9−/− mice to WNV infection correlated with an intact blood-brain barrier since immunoglobulin G, Evans blue leakage into brain, and type IV collagen degradation were markedly reduced in the MMP9−/− mice compared with their levels in controls. Consistent with this, the brain viral loads, selected inflammatory cytokines, and leukocyte infiltrates were significantly reduced in the MMP9−/− mice compared to their levels in wild-type mice. These data suggest that MMP9 plays a role in mediating WNV entry into the central nervous system and that strategies to interrupt this process may influence the course of West Nile encephalitis.

Determining how West Nile virus crosses the blood-brain barrier is critical to understanding the pathogenesis of encephalitis. Here, we show that ICAM-1−/− mice are more resistant than control animals to lethal West Nile encephalitis. ICAM-1−/− mice have a lower viral load, reduced leukocyte infiltration, and diminished neuronal damage in the brain compared to control animals. This is associated with decreased blood-brain barrier leakage after viral infection. These data suggest that ICAM-1 plays an important role in West Nile virus neuroinvasion and that targeting ICAM-1 signaling may help control viral encephalitis.

Lyme arthritis results from colonization of joints by Borrelia burgdorferi and the ensuing host response. Using gene array–based differential analysis of B. burgdorferi gene expression and quantitative reverse trancription-polymerase chain reaction, we identified two paralogous spirochete genes, bmpA and bmpB, that are preferentially up-regulated in mouse joints compared with other organs. Transfer of affinity-purified antibodies against either BmpA or BmpB into B. burgdorferi–infected mice selectively reduced spirochete numbers and inflammation in the joints. B. burgdorferi lacking bmpA/B were therefore generated to further explore the role of these proteins in the pathogenesis of Lyme disease. B. burgdorferi lacking bmpA/B were infectious in mice, but unable to persist in the joints, and they failed to induce severe arthritis. Complementation of the mutant spirochetes with a wild-type copy of the bmpA and bmpB genes partially restored the original phenotype. These data delineate a role for differentially produced B. burgdorferi antigens in spirochete colonization of mouse joints, and suggest new strategies for the treatment of Lyme arthritis.