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.

Summary 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.

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.

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.Mosquito-borne flaviviruses are emerging as increasing threats to human health. For example, West Nile virus (WNV) is maintained worldwide in an enzootic cycle between the avian hosts and mosquito vectors. Since the outbreak in New York City in 1999, WNV has rapidly spread throughout North America, resulting in over 20,000 human cases through 2006, with a mortality rate of 3.9% (11). Infection is often asymptomatic, but high fever, meningitis, encephalitis, or acute flaccid paralysis may occur in 20 to 40% of infected individuals (11). Encephalitis is the major cause of death and is most common in the elderly (Centers for Disease Control and Prevention, Atlanta, GA).The mechanisms by which neurotropic flaviviruses enter the central nervous system (CNS) are not well understood. In theory, WNV can enter the brain through multiple pathways, including endothelial tight junctions, direct infection of endothelial cells, infected leukocytes that traffic to the CNS, infection of olfactory neurons, and/or direct axonal retrograde transport from infected peripheral neurons (7, 27, 28). It has recently been shown that increased peripheral viremia in Tolllike receptor 3 (TLR3)-deficient mice (TLR3 Ϫ/Ϫ ) did not result in increased viral entry into the brain. In contrast, TLR3 Ϫ/Ϫ mice were resistant to lethal WNV challenge owing to better blood-brain barrier (BBB) integrity (30), suggesting that WNV enters the brain primar...

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.