IS-98-ST1 West Nile Virus Derived from an Infectious cDNA Clone Retains Neuroinvasiveness and Neurovirulence Properties of the Original Virus

Infectious clones of West Nile virus (WNV) have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.     

A single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against West Nile virus

BACKGROUND: Lentiviral vectors, due to their capacity to transduce non-dividing cells, have become precious and worldwide used gene transfer systems. Their ability to efficiently and stably transduce dendritic cells (DCs) has led to their successful use as vaccination vectors for eliciting strong, specific and protective cellular immune responses mostly in anti-tumoral but also in anti-viral applications. However, the ability of lentiviral vectors to elicit an antibody-based protective immunity has, to date, not been evaluated. In the present study, we evaluated the potential of a lentiviral vector-based vaccine to elicit humoral immunity against West Nile virus (WNV). WNV is a mosquito-borne flavivirus that emerged in North America and causes encephalitis in humans, birds and horses. Neutralizing anti-WNV antibodies have been shown to be crucial for protection against WNV encephalitis. METHODS: The ability of lentiviral vector TRIP/sE(WNV), expressing the secreted soluble form of the envelope E-glycoprotein (sE(WNV)) from the highly virulent IS-98-ST1 strain of WNV, to induce a specific humoral response and protection against WNV infection was assessed in a mouse model of WNV encephalitis. RESULTS: Remarkably, a single immunization with a minute dose of TRIP/sE(WNV) was efficient at eliciting a long-lasting, protective and sterilizing humoral immunity, only 1 week after priming. CONCLUSIONS: This study broadens the applicability of lentiviral vectors as efficient non-replicating vaccines against pathogens for which a neutralizing humoral response is one active arm of the protective immunity. The TRIP/sE(WNV) lentiviral vector appears to be a promising tool for veterinary vaccination against zoonotic WNV.     

Comparison of the Neuropathology Induced by Two West Nile Virus Strains

Some strains of West Nile virus (WNV) are neuroinvasive and may induce fatal encephalitis/meningitis in a variety of animal species including humans. Whether, however, there is a strain-specific signature in the brain is as yet unknown. Here we investigated the neuropathogenesis induced by two phylogenetically distant WNV strains of lineage 1, WNV_IS98 and WNV_KUN35 911. While four-week old C57Bl/6J mice were susceptible to both strains and succumbed rapidly after intraperitoneal inoculation, differences were observed in virulence and clinical disease. WNV_KUN35 911, the less virulent strain as judged by determination of LD₅₀, induced typical signs of encephalitis. Such signs were not observed in WNV_IS98-infected mice, although they died more rapidly. Histological examination of brain sections also revealed differences, as the level of apoptosis and inflammation was higher in WNV_KUN35 911- than WNV_IS98-infected mice. Moreover, staining for cleaved caspase 3 showed that the two WNV strains induced apoptotic death through different molecular mechanisms in one particular brain area. Finally, the two strains showed similar tropism in cortex, striatum, brainstem, and cerebellum but a different one in hippocampus. In summary, our data show that, upon peripheral administration, WNV_IS98 and WNV_KUN35 911 strains induce partially distinct lesions and tissue tropism in the brain. They suggest that the virulence of a WNV strain is not necessarily correlated with the severity of apoptotic and inflammatory lesions in the brain.     

A Hamster-Derived West Nile Virus Isolate Induces Persistent Renal Infection in Mice

Background

West Nile virus (WNV) can persist long term in the brain and kidney tissues of humans, non-human primates, and hamsters. In this study, mice were infected with WNV strain H8912, previously cultured from the urine of a persistently infected hamster, to determine its pathogenesis in a murine host.

Methodology/Principal Findings

We found that WNV H8912 was highly attenuated for neuroinvasiveness in mice. Following a systemic infection, viral RNA could be detected quickly in blood and spleen and much later in kidneys. WNV H8912 induced constitutive IL-10 production, upregulation of IFN-β and IL-1β expression, and a specific IgM response on day 10 post-infection. WNV H8912 persisted preferentially in kidneys with mild renal inflammation, and less frequently in spleen for up to 2.5 months post infection. This was concurrent with detectable serum WNV-specific IgM and IgG production. There were also significantly fewer WNV- specific T cells and lower inflammatory responses in kidneys than in spleen. Previous studies have shown that systemic wild-type WNV NY99 infection induced virus persistence preferentially in spleen than in mouse kidneys. Here, we noted that splenocytes of WNV H8912-infected mice produced significantly less IL-10 than those of WNV NY99-infected mice. Finally, WNV H8912 was also attenuated in neurovirulence. Following intracranial inoculation, WNV persisted in the brain at a low frequency, concurrent with neither inflammatory responses nor neuronal damage in the brain.

Conclusions

WNV H8912 is highly attenuated in both neuroinvasiveness and neurovirulence in mice. It induces a low and delayed anti-viral response in mice and preferentially persists in the kidneys.     

Infection and injury of neurons by West Nile encephalitis virus

West Nile virus (WNV) infects neurons and leads to encephalitis, paralysis, and death in humans, animals, and birds. We investigated the mechanism by which neuronal injury occurs after WNV infection. Neurons in the anterior horn of the spinal cords of paralyzed mice exhibited a high degree of WNV infection, leukocyte infiltration, and degeneration. Because it was difficult to distinguish whether neuronal injury was caused by viral infection or by the immune system response, a novel tissue culture model for WNV infection was established in neurons derived from embryonic stem (ES) cells. Undifferentiated ES cells were relatively resistant to WNV infection. After differentiation, ES cells expressed neural antigens, acquired a neuronal phenotype, and became permissive for WNV infection. Within 48 h of exposure to an exceedingly low multiplicity of infection (5 x 10(-4)), 50% of ES cell-derived neurons became infected, producing nearly 10(7) PFU of infectious virus per ml, and began to die by an apoptotic mechanism. The establishment of a tractable virus infection model in ES cell-derived neurons facilitates the study of the molecular basis of neurotropism and the mechanisms of viral and immune-mediated neuronal injury after infection by WNV or other neurotropic pathogens.     

Evidence for Co-evolution of West Nile Virus and House Sparrows in North America

West Nile virus (WNV) has been maintained in North America in enzootic cycles between mosquitoes and birds since it was first described in North America in 1999. House sparrows (HOSPs; Passer domesticus) are a highly competent host for WNV that have contributed to the rapid spread of WNV across the U.S.; however, their competence has been evaluated primarily using an early WNV strain (NY99) that is no longer circulating. Herein, we report that the competence of wild HOSPs for the NY99 strain has decreased significantly over time, suggesting that HOSPs may have developed resistance to this early WNV strain. Moreover, recently isolated WNV strains generate higher peak viremias and mortality in contemporary HOSPs compared to NY99. These data indicate that opposing selective pressures in both the virus and avian host have resulted in a net increase in the level of host competence of North American HOSPs for currently circulating WNV strains.     

West Nile virus-induced neuroinflammation: glial infection and capsid protein-mediated neurovirulence

West Nile virus (WNV) infection causes neurological disease at all levels of the neural axis, accompanied by neuroinflammation and neuronal loss, although the underlying mechanisms remain uncertain. Given the substantial activation of neuroinflammatory pathways observed in WNV infection, we hypothesized that WNV-mediated neuroinflammation and cell death occurred through WNV infection of both glia and neurons, which was driven in part by WNV capsid protein expression. Analysis of autopsied neural tissues from humans with WNV encephalomyelitis (WNVE) revealed WNV infection of both neurons and glia. Upregulation of proinflammatory genes, CXCL10, interleukin-1beta, and indolamine-2',3'-deoxygenase with concurrent suppression of the protective astrocyte-specific endoplasmic reticulum stress sensor gene, OASIS (for old astrocyte specifically induced substance), was evident in WNVE patients compared to non-WNVE controls. These findings were supported by increased ex vivo expression of these proinflammatory genes in glia infected by WNV-NY99. WNV infection caused endoplasmic reticulum stress gene induction and apoptosis in neurons but did not affect glial viability. WNV-infected astrocytic cells secreted cytotoxic factors, which caused neuronal apoptosis. The expression of the WNV-NY99 capsid protein in neurons and glia by a Sindbis virus-derived vector (SINrep5-WNVc) caused neuronal death and the release of neurotoxic factors by infected astrocytes, coupled with proinflammatory gene induction and suppression of OASIS. Striatal implantation of SINrep5-WNV(C) induced neuroinflammation in rats, together with the induction of CXCL10 and diminished OASIS expression, compared to controls. Moreover, magnetic resonance neuroimaging showed edema and tissue injury in the vicinity of the SINrep5-WNVc implantation site compared to controls, which was complemented by neurobehavioral abnormalities in the SINrep5-WNVc-implanted animals. These studies underscore the important interactions between the WNV capsid protein and neuroinflammation in the pathogenesis of WNV-induced neurological disorders.     

Recovery and Genetic Characterization of a West Nile Virus Isolate from China

West Nile virus(WNV) is an important neurotropic flavivirus that is widely distributed globally. WNV strain XJ11129 was first isolated in Xinjiang, China, and its genetic and biological characteristics remain largely unknown. In this study,phylogenetic and sequence analyses revealed that XJ11129 belongs to lineage 1 a and shares high genetic identity with the highly pathogenic strain NY99. Then, the full-length genomic c DNA of XJ11129 was amplified and assembled using a modified Gibson assembly(GA) method. The virus(named r XJ11129) was successfully rescued in days following this method. Compared with other wild-type WNV isolates, r XJ11129 exhibited virulence indistinguishable from that of the NY99 strain in vivo. In summary, the genomic and virulence phenotypes of r XJ11129 were characterized in vivo and in vitro, and these data will improve the understanding of the spread and pathogenesis of this reemerging virus.     

Persistence of West Nile virus

West Nile virus (WNV) is a widespread global pathogen that results in significant morbidity and mortality. Data from animal models provide evidence of persistent renal and neurological infection from WNV; however, the possibility of persistent infection in humans and long-term neurological and renal outcomes related to viral persistence remain largely unknown. In this paper, we provide a review of the literature related to persistent infection in parallel with the findings from cohorts of patients with a history of WNV infection. The next steps for enhancing our understanding of WNV as a persistent pathogen are discussed.     

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.     

West Nile fever/encephalitis

West Nile virus (WNV), a member of the family Flaviviridae (genus Flavivirus), is a mosquito-borne virus first isolated in 1937 in the West Nile district of Uganda. The disease in humans is characterized by a dengue-like illness with fever, and a more severe form is characterized by central nervous system involvement, including encephalitis, meningitis, and myelitis. WN encephalitis was first reported in the Western Hemisphere in the summer of 1999, there was an outbreak in New York City. Epidemic WNV strains in North America are severely pathogenic, however, attenuated WNV strains were found in Texas and Mexico in 2003. The principal vectors of WNV transmission in North America are Culex. pipiens, Cx. Quinquefasciatus, Cx. restuans, Cx salinarius and Cx talsalis. The number of WN fever case has exceeded 27,000 since 1999 in the United States and 4,600 since 2002 in Canada. The first imported case of West Nile fever in Japan was confirmed in September, 2005. The patient had returned to Japan from the United States and developed symptoms the next day. There is currently no WN vaccine for use in humans. An inactivated WNV vaccine for use in horses has been available since 2001. A DNA vaccine, a chimeric live attenuated vaccine, and a recombinant vaccine have also been licensed for use in horses.     

实验感染的三带喙库蚊和来亨鸡体内西尼罗病毒的分离与鉴定

采用C6/36细胞培养分离活病毒、间接免疫荧光染色检测病毒抗原、RT-PCR扩增病毒基因片段和PCR产物测序等方法,对实验感染的三带喙库蚊Culex tritaeniorhynchus和来亨鸡血液样本中的西尼罗病毒进行分离和鉴定。结果表明,接种实验感染蚊虫研磨液和来亨鸡血液样本的C6/36细胞出现细胞融合、空泡形成的病变效应; 用西尼罗病毒抗血清进行间接免疫荧光染色,感染病毒的细胞呈现黄绿色荧光,为阳性反应; 采用3对不同引物的RT- PCR体系扩增分别出现预期的408 bp、498 bp和559 bp的基因片段,序列测定证实扩增序列与实验所用毒株相应的基因序列基本相同。从而证实实验感染三带喙库蚊和来亨鸡体血液内的西尼罗病毒与实验感染所用的西尼罗病毒Chin-01株一致。     

西尼罗病毒易感性研究现状

西尼罗病毒可引起鼠、人类及其他动物严重脑炎,近年来再度流行。基因研究分析表明近年来西尼罗病毒突变的速度逐渐加快,突变主要发生在基因编码区,这些突变可导致病毒蛋白E区、NS1区、NS2区及NS5区氨基酸序列的改变。西尼罗病毒感染主要发生于老龄及免疫缺陷动物或人群。西尼罗病毒的易感染性除与突变有重要关系外,与CCR5、OAS及CXCL10等受体及因子也有一定的关系。     

West Nile virus core protein; tetramer structure and ribbon formation

We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four alpha helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked alpha helices seen in HEAT protein structures.     

Identification of novel small-molecule inhibitors of West Nile virus infection

West Nile virus (WNV) has spread throughout the United States and Canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. No therapy or vaccine is currently approved for use in humans. Using high-throughput screening assays that included a luciferase expressing WNV subgenomic replicon and an NS1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,000 compounds for their capacity to inhibit WNV replication. We identified 10 compounds with strong inhibitory activity against genetically diverse WNV and Kunjin virus isolates. Many of the inhibitory compounds belonged to a chemical family of secondary sulfonamides and have not been described previously to inhibit WNV or other related or unrelated viruses. Several of these compounds inhibited WNV infection in the submicromolar range, had selectivity indices of greater than 10, and inhibited replication of other flaviviruses, including dengue and yellow fever viruses. One of the most promising compounds, AP30451, specifically blocked translation of a yellow fever virus replicon but not a Sindbis virus replicon or an internal ribosome entry site containing mRNA. Overall, these compounds comprise a novel class of promising inhibitors for therapy against WNV and other flavivirus infections in humans.     

Immunochemical properties of short recombinant polypeptides of proteine of, the West Nile virus bearing epitopes of domains I and II

Fragments cDNA (nt 935-1475, 1091-1310, 935-1193) encoding N-terminal part of protein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human were obtained and cloned. Recombinant polypeptides of glycoprotein E (E1-86, E53-126, E1-180) of the WNV with corresponding amino acid sequence to the cloned fragments of cDNA and modeling the epitopes of domains I and II of surface glycoprotein E were purified by affinity chromatography. Twelve types of monoclonal antibodies (MAbs) created in our laboratory against recombinant polypeptide E1-180 interact with glycoprotein E of the WNV as results of Western blot and ELISA that is demonstrating an similarity of chemical structure of short recombinant polypeptides and corresponding amino acid sequence regions of WNV protein E. Analysis of interactions of MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus let us reveal no less than six epitopes within domains I and II of glycoprotein E of the WNV. No less than seven types of MAbs to 86-126 aa region of the domain II were found where located peptide providing fusion of virus--cell membranes (98-110 aa). The epitope for anti-receptor MAbs 10H10 within 53-86 aa region of domain II of protein E of the WNV was mapped and it shows that the fusion peptide and co-receptor of protein E for cellular laminin-binding protein (LBP) are spatial nearness. X-ray model of protein E let us suppose that bc-loop (73-89 aa) of domain II interacts with LBP and together with cd-loop (fusion peptide) determines an initial stages of penetration virions into cell.     

西尼罗病毒研究进展

西尼罗病毒（West Nile virus,WNV）属黄病毒科,为正单链RNA病毒。它在人类中的感染导致以发热为主要症状的传染性疾病,主要由蚊虫叮咬传播。自20世纪50年代首例报告西尼罗病毒自然感染所致脑炎后的几十年内,西尼罗病毒脑炎在欧洲及中亚地区散在、小规模流行。西尼罗病毒脑炎于1999年在美国的爆发及随后几年在北美的流行引起了极大的关注。这次爆发流行中新出现的种种迹象,如其中间宿主——野生鸟类的大量死亡,人类感染者中中枢神经系统受损比例的增高等,提示近期的遗传变异已使西尼罗病毒感染的病理学与流行病学发生了较显著的变化。另外,随着感染的流行,蚊虫叮咬以外的传播途径,如输血、器官移植、母婴传播等日益受到人们重视。同时,人们对阻止疫情所急需的疫苗的研制也在进行之中。本文就近几年来对西尼罗病毒的感染、免疫与流行病学方面的研究进展进行了综述。     

Caspase 3-dependent cell death of neurons contributes to the pathogenesis of West Nile virus encephalitis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3(-/-) mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3(-/-) mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.     

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3(-/-)) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3(-/-) mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-alpha receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.