一株西尼罗病毒株的生物学特征研究

目的对引进的西尼罗病毒(WNV)毒株的形态学、致病性、细胞敏感性、免疫原性等生物学性状进行探讨，为进一步开展WNV的相关研究奠定基础。方法选择Vero-E6与C6／36细胞观察WNV的细胞病变效应(CPE)，并制作电镜负染标本和组织切片标本进行病毒形态学鉴定；通过乳鼠脑内接种WNV确认其致病性；以灭活的感染乳鼠脑悬液免疫BALB／c小鼠，以间接免疫荧光试验(IFA)测定其血清WNV IgG抗体；用RT-PCR检测病毒核酸，扩增的核苷酸序列通过BLAST作同源性比较。结果WNV所致Vero-E6与C6／36细胞的CPE分别以细胞圆缩和融合为主要特征；电镜下所见病毒体为有包膜、直径约30～50nm的球形颗粒；脑内接种WNV可致全部乳鼠死亡；灭活病毒可诱导小鼠产生WNV抗体；经RT-PCR扩增，在WNV培养液及感染乳鼠脑组织中均检测到目的基因片段，且仅与WNV有较高的同源性(94％～100％)。结论本研究使用的WNV毒株可供进一步开展WNV的相关研究。     

Fatal fulminant pan-meningo-polioencephalitis due to West Nile virus

We report a case of fatal fulminant West Nile virus (WNV) meningoencephalitis in an 87-year-old white male gardener. The Pennsylvania patient presented with a 3-day history of flu-like symptoms. His hospital course was gravely precipitous with onset of coma, ventilator dependence, loss of cortical and brainstem functions within ten days of admission. Acute serum and cerebrospinal fluid samples revealed elevated levels of WNV IgM antibodies by ELISA as well as elevated CSF white blood cells, protein and glucose. A complete autopsy revealed a multifocal lymphocytic myocarditis and severe chronic tubulointerstitial nephritis. Viral culture and PCR analysis of post-mortem samples of the spleen, kidney and brain were positive for WNV. Histological sections from all regions of the brain and spinal cord demonstrated a severe, non-necrotizing, subacute, polio-meningoencephalitis. While both gray and white matter were inflamed, gray matter was much more severely involved. Many gray matter nuclei showed severe neuronal loss with residual dying neurons surrounded by activated microglia. Immunohistochemical stains revealed profuse infiltration of the meninges and cerebral parenchyma by CD8 T-lymphocytes and perivascular B-lymphocytes. Electron micrographs revealed diffuse intracellular and extracellular edema but no viral particles were identified. Immunohistochemical and immunofluorescent staining for WNV filled the cytoplasm of residual neurons. West Nile virus mediates a predominantly polioencephalitis secondary to direct infection of neurons.     

Clinicopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis

The differences in pathologic findings of fatal cases of West Nile virus (WNV) encephalitis in the context of underlying conditions and illness duration are not well known. During 2002, we studied central nervous system (CNS) tissue samples from 23 patients who had serologic and immunohistochemical (IHC) evidence of a recent WNV infection. Fifteen patients had underlying medical conditions (5 malignancies, 3 renal transplants, 3 with diabetes or on dialysis, 2 with AIDS, and 2 receiving steroids). WNV serology was positive for 18 patients, negative for 2, and not available for 3. Perivascular lymphocytic infiltrates, microglial nodules, and loss of neurons were predominantly observed in the brainstem and anterior horns in the spinal cord. IHC using antibodies against flaviviruses and WNV showed viral antigens in 12 (52%) of 23 patients. Viral antigens were found inside neurons and neuronal processes predominantly in the brainstem and anterior horns. In general, the antigens were focal and sparse; however, in 4 severely immunosuppressed patients, extensive viral antigens were seen throughout the CNS. Positive IHC staining was observed in tissues of 7 of 8 patients who died within 1 week after illness onset, compared with 4 of 14 with more than 2 weeks' illness duration. WNV causes an encephalomyelitis by primarily affecting brainstem and spinal cord. Differences in the amount of viral antigen may be related to underlying medical conditions and length of survival. IHC can be an important diagnostic method, particularly during the 1st week of illness, when antigen levels are high.     

Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action

Flaviviruses are emerging threats to public health worldwide. Recently, one flavivirus, West Nile virus (WNV), has caused the largest epidemic of viral encephalitis in US history. Like other flaviviruses, WNV is thought to cause a persistent infection in insect cells, but an acute cytopathic infection of mammalian cells. To study adaptation of WNV to persistently replicate in cell culture and generate a system capable of detecting antiviral compounds in the absence of live virus, we generated subgenomic replicons of WNV and adapted these to persistently replicate in mammalian cells. Here we report that adaptation of these replicons to cell culture results in a reduction of genome copy number, and demonstrate that hamster, monkey, and human cells that stably carry the replicons can be used as surrogates to detect the activity of anti-WNV compounds. Additionally, we have used these cells to investigate the interaction of WNV genomes with interferon (IFN). These studies demonstrated that IFN can cure cells of replicons and that replicon-bearing cells display lower responses to IFN than their IFN-cured derivatives.     

West Nile Virus Encephalitis 16 Years Later

Arboviruses (Arthropod‐borne viruses) include several families of viruses (Flaviviridae, Togaviradae, Bunyaviradae, Reoviradae) that are spread by arthropod vectors, most commonly mosquitoes, ticks and sandflies. The RNA genome allows these viruses to rapidly adapt to ever‐changing host and environmental conditions. Thus, these virus families are largely responsible for the recent expansion in geographic range of emerging viruses including West Nile virus (WNV), dengue virus and Chikungunya virus. This review will focus on WNV, especially as it has progressively spread westward in North America since its introduction in New York in 1999. By 2003, WNV infections in humans had reached almost all lower 48 contiguous United States (US) and since that time, fluctuations in outbreaks have occurred. Cases decreased between 2008 and 2011, followed by a dramatic flair in 2012, with the epicenter in the Dallas‐Fort Worth region of Texas. The 2012 outbreak was associated with an increase in reported neuroinvasive cases. Neuroinvasive disease continues to be a problem particularly in the elderly and immunocompromised populations, although WNV infections also represented the second most frequent cause of pediatric encephalitis in these same years. Neuropathological features in cases from the 2012 epidemic highlight the extent of viral damage that can occur in the CNS.     

Reversal of West Nile virus-induced blood-brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor

Though compromised blood-brain barrier (BBB) is a pathological hallmark of WNV-associated neurological sequelae, underlying mechanisms are unclear. We characterized the expression of matrix metalloproteinases (MMP) in WNV-infected human brain microvascular endothelial cells (HBMVE) and human brain cortical astrocytes (HBCA), components of BBB and their role in BBB disruption. Expression of multiple MMPs was significantly induced in WNV-infected HBCA cells. Naïve HBMVE cells incubated with the supernatant from WNV-infected HBCA cells demonstrated loss of tight junction proteins, which were rescued in the presence of MMP inhibitor, GM6001. Further, supernatant from WNV-infected HBCA cells compromised the in vitro BBB model integrity. Our data suggest astrocytes as one of the sources of MMP in the brain, which mediates BBB disruption allowing unrestricted entry of immune cells into the brain, thereby contributing to WNV neuropathogenesis. Because of the unavailability of WNV antivirals and vaccines, use of MMP inhibitors as an adjunct therapy to ameliorate WNV disease progression is warranted.     

Development of Monoclonal Antibodies to West Nile Virus and Their Application in Immunohistochemistry

West Nile virus (WNV) is endemic throughout Africa, Eurasia, America, and Australia and has important implications for avian, horse, and human health. In these regions, dead birds are monitored for the presence of WNV through immunohistochemistry (IHC) and PCR. However, a number of the tools for IHC are inadequate owing to their cross-reactivity to other Japanese encephalitis serogroup viruses. Here we have established eight monoclonal antibodies (MAbs) to WNV. Four of them bound to the envelope protein, three of them bound to nonstructural protein 1 (NS1), and one bound to precursor membrane protein (prM), as shown by Western blot analysis. The anti-NS1 MAbs and the anti-prM MAb did not cross-react with Japanese encephalitis virus (JEV), Murray valley encephalitis virus, or St. Louis encephalitis virus in an indirect enzyme-linked immunosorbent assay. One NS1-specific MAb, SHW-32B1, and the previously reported NS1-specific MAb, SHW-7A11, were shown by IHC to specifically detect the cytoplasm of degenerated cells in the heart and brain of a WNV-infected goose. Neither of these MAbs were shown by IHC to cross-react with degenerated cells in the brain of a JEV-infected pig. These MAbs are the first reported anti-NS1 MAbs that can be used for WNV-specific IHC using formalin-fixed, paraffin-embedded sections. They may be useful for WNV research and surveillance.     

Isolation of West Nile Virus from Urine Samples of Patients with Acute Infection

This study demonstrated that West Nile virus (WNV) excreted in the urine of patients with acute infection can be isolated in cell cultures. In addition, the protocols for WNV isolation from urine samples were standardized, and factors that may affect the efficiency of WNV isolation were identified.     

西尼罗病毒与乙型脑炎病毒生物学性状的比较与鉴别

目的对西尼罗病毒(WNV)和乙脑病毒(JEV)的致细胞病变效应(CPE)、致病性、形态学、免疫学和分子生物学性状进行比较,为WNV感染的鉴别诊断提供依据。方法通过给C6/36细胞和乳鼠接种WNV或JEV,观察两种病毒的CPE和致病性;制作组织切片的透射电镜标本进行病毒形态学观察;分别用间接免疫荧光试验(IFA)和RTPCR方法对两种病毒抗体阳性血清及病毒核酸进行检测。结果WNV和JEV所致C6/36细胞CPE分别以细胞融合和细胞脱落为主要特征;脑内接种两种病毒对乳鼠的致病性未见明显差别;电镜下,两种病毒体的形态与大小相似;WNV与JEV之间存在抗原交叉反应;用黄病毒通用引物可从WNV和JEV感染组织中检出病毒核酸,而用特异引物仅能扩增出相应病毒的核苷酸片段。结论病毒所致C6/36细胞CPE与病毒核酸检测可作为WNV与JEV有效的鉴别方法,对两种病毒抗体效价进行同时测定有助于病原体感染的诊断。     

实时RT-PCR检测西尼罗病毒

目的建立西尼罗病毒快速、敏感和特异的实时RT-PCR法,用于西尼罗病毒疾病的早期诊断。方法对Vero细胞增殖的西尼罗病毒分别进行常规RT-PCR和实时RT-PCR法扩增,以PFU／ml为参考标准,比较二者的敏感性和特异性。并用实时RT-PCR法检测西尼罗病毒感染的小鼠组织标本。结果采用所建立的实时RT-PCR法和常规RT-PCR法可分别检测到0．02PFU／ml和2PFU／ml的西尼罗病毒RNA,前者的敏感性比后者高100倍。而对其他黄病毒科成员的检测均为阴性,表明该方法是特异的。采用这种实时RT-PCR法可从西尼罗病毒感染小鼠的血液、脾脏、肾脏和脑组织标本中检测出病毒核酸,且在感染后第2天最先自血液和脾脏中检测到病毒,在感染后1周的脑组织中的病毒滴度最高。结论本研究建立的实时RT-PCR法具有很高的敏感性和特异性,因此该方法可用于西尼罗病毒疾病的早期监测和流行病学研究。     

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.     

Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death

Background  

WNV-associated encephalitis (WNVE) is characterized by increased production of pro-inflammatory mediators, glial cells activation and eventual loss of neurons. WNV infection of neurons is rapidly progressive and destructive whereas infection of non-neuronal brain cells is limited. However, the role of neurons and pathological consequences of pro-inflammatory cytokines released as a result of WNV infection is unclear. Therefore, the objective of this study was to examine the role of key cytokines secreted by WNV-infected neurons in mediating neuroinflammatory markers and neuronal death.     

Systemic distribution of West Nile virus infection: postmortem immunohistochemical study of six cases

Rare cases of West Nile virus (WNV)-associated inflammation outside the central nervous system (CNS) have been reported. We evaluated the systemic distribution of WNV in postmortem tissues during encephalitis in six patients using immunohistochemistry. WNV antigens were detected in neurons of CNS (all 6 cases), kidney (4 cases), lungs (2 cases), pancreas (2 cases), thyroid (2 cases), intestine (2 cases), stomach (1 case), esophagus (1 case), bile duct (1 case), skin (1 case), prostate (1 case) and testis (1 case). In systemic organs epithelial cells were infected. In none of the six cases were viral antigens identified in hepatocytes, heart, adrenal gland, nerves, skeletal muscles, bone, vessels and fat. All cases in which viral antigens were identified in systemic organs in addition to CNS were severely immunocompromised transplant recipients. With the exception of testis and brain, most foci of infection were not associated with inflammation. While the absence of inflammation may in part be due to patient immunosuppression or to possible transient nature of any host response, compartmentalization of viral antigen to the luminal region of epithelial cells may sequester WNV from immune recognition. Comparison of our findings with previous reports suggests that patients with WNV encephalitis can have widespread systemic infection.     

Enzyme-Linked Immunosorbent Assays Using Recombinant Envelope Protein Expressed in COS-1 and Drosophila S2 Cells for Detection of West Nile Virus Immunoglobulin M in Serum or Cerebrospinal Fluid

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.     

An infectious West Nile virus that expresses a GFP reporter gene

West Nile virus is a mosquito-borne, neurotropic flavivirus that causes encephalitis in humans and animals. Since being introduced into the Western hemisphere in 1999, WNV has spread rapidly across North America, identifying this virus as an important emerging pathogen. In this study, we developed a DNA-launched infectious molecular clone of WNV that encodes a GFP reporter gene. Transfection of cells with the plasmid encoding this recombinant virus (pWNII-GFP) resulted in the production of infectious WNV capable of expressing GFP at high levels shortly after infection of a variety of cell types, including primary neurons and dendritic cells. Infection of cells with WNII-GFP virus was productive, and could be inhibited with both monoclonal antibodies and interferon-beta, highlighting the potential of this system in the development and characterization of novel inhibitors and therapeutics for WNV infection. As expected, insertion of the reporter gene into the viral genome was associated with a reduced rate of viral replication, providing the selective pressure for the development of variants that no longer encoded the full-length reporter gene cassette. We anticipate this DNA-based, infectious WNV reporter virus will allow novel approaches for the study of WNV infection and its inhibition both in vitro and in vivo.     

West Nile virus infection of the placenta

Intrauterine infection of fetuses with West Nile virus (WNV) has been implicated in cases of women infected during pregnancy. Infection of timed-pregnant mice on 5.5, 7.5, and 9.5 days post-coitus (dpc) resulted in fetal infection. Infection of dams on 11.5 and 14.5 dpc resulted in little and no fetal infection, respectively. Pre-implantation embryos in culture were also infected with WNV after the blastocyst stage and the formation of trophectoderm. Green fluorescent protein (GFP) expression was observed in a trophoblast stem (TS) cell line after infection with a GFP-expressing WNV construct. However, no fluorescence was observed in differentiated trophoblast giant cell (TGC) cultures. GFP fluorescence was present in TGC cultures if infected TS cells were induced to differentiate. These results suggest that embryos are susceptible to WNV infection after the formation of the trophectoderm around 3.5 dpc through the formation of the functional placenta around 10.5 dpc.     

Ultrastructural study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus (Diptera: Culicidae)

The ultrastructural features of West Nile virus (WNV) replication and dissemination in orally infected Culex pipiens quinquefasciatus Say were analyzed over a 25-d infection period. To investigate the effects of virus replication on membrane induction, cellular organization, and cell viability in midgut and salivary gland tissues, midguts were dissected on days 3, 7, 14, and 21, and salivary glands were collected on days 7, 14, 21, and 25 postinfection (d.p.i.) for examination by transmission electron microscopy (TEM). Whole mosquito heads were embedded for TEM analysis 14 d.p.i. to localize WNV particles and to investigate the effects of replication on nervous tissues of the brain. Membrane proliferation was induced by WNV in the midgut epithelium, midgut muscles, and salivary glands, although extensive endoplasmic reticulum swelling was a unique feature of salivary gland infection. TEM revealed WNV-induced pathology in salivary glands at 14, 21, and 25 d.p.i., and we hypothesize that long-term virus infection of this tissue results in severe cellular degeneration and apoptotic-like cell death. This finding indicates that the efficiency of WNV transmission may decrease with mosquito age postinfection.     

West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier

Neurological complications such as inflammation, failure of the blood-brain barrier (BBB), and neuronal death contribute to the mortality and morbidity associated with WNV-induced meningitis. Compromised BBB indicates the ability of the virus to gain entry into the CNS via the BBB, however, the underlying mechanisms, and the specific cell types associated with WNV-CNS trafficking are not well understood. Brain microvascular endothelial cells, the main component of the BBB, represent a barrier to virus dissemination into the CNS and could play key role in WNV spread via hematogenous route. To investigate WNV entry into the CNS, we infected primary human brain microvascular endothelial (HBMVE) cells with the neurovirulent strain of WNV (NY99) and examined WNV replication kinetics together with the changes in the expressions of key tight junction proteins (TJP) and cell adhesion molecules (CAM). WNV infection of HBMVE cells was productive as analyzed by plaque assay and qRT-PCR, and did not induce cytopathic effect. Increased mRNA and protein expressions of TJP (claudin-1) and CAM (vascular cell adhesion molecule and E-selectin) were observed at days 2 and 3 after infection, respectively, which coincided with the peak in WNV replication. Further, using an in vitro BBB model comprised of HBMVE cells, we demonstrate that cell-free WNV can cross the BBB, without compromising the BBB integrity. These data suggest that infection of HBMVE cells can facilitate entry of cell-free virus into the CNS without disturbing the BBB, and increased CAM may assist in the trafficking of WNV-infected immune cells into the CNS, via ‘Trojan horse’ mechanism, thereby contributing to WNV dissemination in the CNS and associated pathology.