Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection

The domain III of the West Nile virus (WNV) envelope glycoprotein (E) was shown to serve as virus attachment domain to the cellular receptor, and neutralizing Abs have been mapped to this specific domain. In this study, domain III of the WNV E protein (WNV E DIII) was expressed as a recombinant protein and its potential as a subunit vaccine candidate was evaluated in BALB/C mice. Immunization of WNV E DIII protein with oligodeoxynucleotides (CpG-DNA) adjuvant by i.p. injection was conducted over a period of 3 wk. The immunized mice generated high titer of WNV-neutralizing Abs. Murine Ab against WNV E DIII protein was also capable of neutralizing Japanese encephalitis virus. The IgG isotypes generated were predominantly IgG2a in the murine sera against the recombinant protein. Splenocyte cultures from the mice coadministrated with WNV E DIII protein and CpG secreted large amounts of IFN-gamma and IL-2 and showed proliferation of T cells in the presence of WNV E DIII protein. Overall, this study highlighted that recombinant WNV E DIII protein delivered in combination with CpG adjuvant to mice generated a Th1 immune response type against WNV and can serve as a potential vaccine to prevent WNV infection.     

Immunization of mice against West Nile virus with recombinant envelope protein.

West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis. Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli. The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments. Patients with WN virus infection had Abs that recognized the recombinant E protein. C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus. Passive administration of E protein antisera was also sufficient to afford immunity. E protein is a candidate vaccine to prevent WN virus infection.     

A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5) TCID(50) Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.     

Immunological headgear: antiviral immune responses protect against neuroinvasive West Nile virus

With the emergence of epidemic strains of West Nile virus (WNV) in North America, there has been a surge in new research and knowledge regarding the peripheral immune responses that prevent neuroinvasion, the routes of WNV entry into the central nervous system (CNS) and the critical CNS immune responses that promote viral clearance and recovery at this anatomic site. WNV infection induces archetypal antiviral immune responses that, in most cases, lead to elimination of the virus with relatively few immunopathological consequences. Here, we present our current understanding of the innate and adaptive immune responses that limit dissemination to the CNS from WNV infection and the antiviral immune responses within the CNS that intervene when they fail.     

Crystal structure of the West Nile virus envelope glycoprotein

The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-A crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel alpha-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure.     

Innate immune control of West Nile virus infection

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 signalling 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.     

Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein

Using a panel of neutralizing monoclonal antibodies, we have mapped epitopes in domain III of the envelope protein of the New York strain of West Nile virus. The ability of monoclonal antibodies that recognize these epitopes to neutralize virus appeared to differ between lineage I and II West Nile virus strains, and epitopes were located on the upper surface of domain III at residues E307, E330, and E332.     

Innate immune evasion by hepatitis C virus and West Nile virus

Antiviral immunity in mammals involves several levels of surveillance and effector actions by host factors to detect viral pathogens, trigger /β interferon production, and to mediate innate defenses within infected cells. Our studies have focused on understanding how these processes are regulated during infection by hepatitis C virus (HCV) and West Nile virus (WNV). Both viruses are members of the Flaviviridae and are human pathogens, but they each mediate a very different disease and course of infection. Our results demonstrate common and unique innate immune interactions of each virus that govern antiviral immunity and demonstrate the central role of /β interferon immune defenses in controlling the outcome of infection.     

Evasion and disruption of innate immune signalling by hepatitis C and West Nile viruses

Signalling pathways leading to type I interferon production are the first line of defence employed by the host to combat viruses, and represent a barrier that an invading virus must overcome in order to establish infection. In this review we highlight the ability of two members of the Flaviviridae, a globally distributed family of RNA viruses that represent a significant public health concern, to disrupt and evade these defences. Hepatitis C virus is a hepatotropic virus, infecting greater than 170 million people worldwide, while West Nile virus is a neurotropic virus that causes encephalitis in humans and horses. While these viruses cause distinct disease phenotypes, the ability of pathogenic strains to modulate the innate immune response is a key factor in influencing disease outcome. Both viruses have evolved unique strategies to target various aspects of type I interferon induction and signalling in order to prevent viral clearance and to promote virus replication.     

Cholesterol manipulation by West Nile virus perturbs the cellular immune response

Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically hampered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.     

非洲猪瘟病毒I226R蛋白抑制cGAS-STING通路介导的天然免疫应答

本研究旨在探究非洲猪瘟病毒(African swine fever virus, ASFV) I226R蛋白(I226R protein, pI226R)抑制cGAS-STING信号通路的作用机制。利用双荧光素酶报告系统和实时荧光定量PCR (real-time quantitative PCR, qPCR)证明pI226R显著抑制cGAS-STING通路介导的I型干扰素及干扰素刺激相关基因的产生。免疫共沉淀及激光共聚焦显微镜试验发现pI226R与cGAS蛋白相互作用。免疫印迹分析证明pI226R通过自噬-溶酶体途径促进cGAS蛋白的降解。同时,pI226R阻碍了cGAS与E3泛素连接酶三基序蛋白56 (tripartite motif protein 56, TRIM56)的结合,导致cGAS的单泛素化减弱,从而抑制了cGAS的活化和cGAS-STING通路的激活。总之,本研究证明ASFV pI226R通过拮抗cGAS进而抑制宿主的抗病毒天然免疫反应,进一步增加了对研究ASFV免疫逃逸机制的理解,为疫苗的研发提供了理论基础。     

Junin virus infects mouse cells and induces innate immune responses

Junín virus is the causative agent for Argentine hemorrhagic fever, and its natural host is the New World rodent Calomys musculinus. The virus is transmitted to humans by aerosolization, and it is believed that many of the clinical symptoms are caused by cytokines produced by sentinel cells of the immune system. Here we used the Junín virus vaccine strain Candid 1 to determine whether mouse cells could be used to study virus entry and antiviral innate immune responses. We show that Candid 1 can infect and propagate in different mouse-derived cell lines through a low-pH-dependent, transferrin receptor 1-independent mechanism, suggesting that there is a second entry receptor. In addition, Candid 1 induced expression of the antiviral cytokines tumor necrosis factor alpha and beta interferon in macrophages, and this induction was independent of viral replication. Using Candid 1, as well as virus-like particles bearing the viral glycoprotein, to infect different primary cells and established macrophage cell lines with deletions in the Toll-like receptor (TLR) pathway, we show that TLR2 is a cellular sensor of both the Parodi and Candid 1 viral glycoproteins. Because Junín virus is highly lethal in humans, the use of an experimentally tractable model system, such as the mouse, could provide a better understanding of the antiviral innate cellular responses to Junín virus and the role of these responses in pathogenesis.