Jianfeng Dai

Matrix Metalloproteinase 9 Facilitates West Nile Virus Entry into the Brain

ABSTRACT 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.

A Paradoxical Role for Neutrophils in the Pathogenesis of West Nile Virus

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 PMN-attracting 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.

A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes

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 homolog 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.

Antibodies against a Tick Protein, Salp15, Protect Mice from the Lyme Disease Agent

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.

Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I

Caspase-12 has been shown to negatively modulate inflammasome signaling during bacterial infection. Its function in viral immunity, however, has not been characterized. We now report an important role for caspase-12 in controlling viral infection via the pattern-recognition receptor RIG-I. After challenge with West Nile virus (WNV), caspase-12-deficient mice had greater mortality, higher viral burden and defective type I interferon response compared with those of challenged wild-type mice. In vitro studies of primary neurons and mouse embryonic fibroblasts showed that caspase-12 positively modulated the production of type I interferon by regulating E3 ubiquitin ligase TRIM25–mediated ubiquitination of RIG-I, a critical signaling event for the type I interferon response to WNV and other important viral pathogens.

ICAM-1 Participates in the Entry of West Nile Virus into the Central Nervous System

ABSTRACT 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.

Tick Histamine Release Factor Is Critical for Ixodes scapularis Engorgement and Transmission of the Lyme Disease Agent

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.

ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro

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

IL-22 Signaling Contributes to West Nile Encephalitis Pathogenesis

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.

Molecular interactions that enable movement of the Lyme disease agent from the tick gut into the hemolymph.

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)2 fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.