Detection of West Nile virus lineage 2 in mosquitoes during a human outbreak in Greece

A human outbreak of West Nile virus (WNV) infections occurred in 2010 in central Macedonia, northern Greece. Most cases were observed close to four rivers forming a large Delta, a major Mediterranean wetland. WNV lineage 2 sequences were obtained from two pools of Culex pipiens mosquitoes trapped in sites where encephalitis cases occurred a few days before the trapping. The Greek strain showed the highest homology to Hungarian and South African strains, differing from the Russian WNV lineage 2 strain, which suggests that at least two lineage 2 strains have been introduced and established in Europe, causing severe disease to humans.     

Circulation of West Nile virus lineage 1 and 2 during an outbreak in Italy

In 2011, from 26 September to 16 October, a small outbreak of West Nile virus (WNV) disease occurred on the island of Sardinia (Italy). According to the national case definition, six cases with acute neurological disease were confirmed in hospitalized patients, and four of them died; one of these was only 34 years old. In two case, WNV RNA was detected in urine, suggesting renal involvement. Sequence analysis showed lineage 1 and 2 circulation.     

Non-neuroinvasive West Nile virus infections during the outbreak in Greece

A major outbreak of West Nile virus (WNV) infections took place in 2010 in Greece. Apart from the neuroinvasive cases, many additional cases without involvement of the nervous system were observed, characterized by high fever, myalgia, rash, leukopenia, and long-lasting recovery. West Nile nonneuroinvasive disease is a distinct clinical syndrome, and is not always mild.     

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。     

Persistence of West Nile virus immunoglobulin M antibodies, Greece

A major outbreak of West Nile virus (WNV) lineage 2 infections was observed in 2010 in Greece. In order to check the persistence of WNV IgM antibodies, a second serum sample taken 75-180 days after onset of the illness from 29 patients with WNV infection was tested. A third sample was obtained 181-270 days after onset of the illness from 8 of the 12 patients with IgM-positive second sample. Mixed effects linear regression analysis indicated that the approximate time at which IgM index became negative was 164 (95% confidence interval, 95% CI 99-236) days after the symptoms' onset. Persistence of IgM antibodies was observed in 12% of patients at 181-270 days of follow-up. A sharp decrease in the IgM levels was observed, mainly in patients who had high IgM index value in the acute phase. All patients were WNV IgG positive at the follow-up.     

Acute West Nile virus neuroinvasive infections: cross-reactivity with dengue virus and tick-borne encephalitis virus

Cross-reactions in serology are common among flaviviruses. During the outbreak of West Nile virus (WNV) infections in Greece in 2010, WNV IgM-positive serum and cerebrospinal fluid samples were tested for the presence of IgM and IgG antibodies against Dengue virus (DENV) and tick-borne encephalitis virus. Higher cross-reactivity was observed in IgM antibodies between WNV and DENV; however, the index of the WNV antibodies was in all cases higher than that of the DENV antibodies. There is a need for caution when evaluating serologic results of flaviviral infections, while efforts have to be focused on the development of diagnostic assays with increased specificity.     

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。     

Novel West Nile virus lineage 1a full genome sequences from human cases of infection in north-eastern Italy, 2011

During 2008–2009, several human cases of WNV disease caused by an endemic lineage 1a strain were reported in areas surrounding the Po river in north-eastern Italy. Since 2010, cases have been recorded in nearby northern areas, where, in 2011, both lineage 1a and 2 were detected. We describe here two new WNV complete genome sequences from human cases of WNV infection occurring in 2011 in the Veneto Region. Phylogenetic analysis showed that both genome sequences belonged to lineage 1a and were related to WNV strains of the Western Mediterranean subtype. The novel WNV genomes had high nucleotide and amino acid sequence divergence from each other and from the WNV strain circulating in Italy in 2008–2009. The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease that requires further investigation.     

The First Reported Case of West Nile Encephalitis in Korea

West Nile encephalitis was first identified in 1937, but until now, it was never diagnosed in Korea. A 58-yr-old Korean man was admitted with headache and cognitive dysfunction. The patient had been on a business trip in Guinea. Cerebrospinal fluid (CSF) showed pleocytosis. The patient complained of both leg weakness,and arachnoiditis and myelitis were observed on lumbar magnetic resonance imaging (MRI). A specific neutralizing antibody for West Nile virus was positive in serum. After a treatment with interferon-α 3mu, follow up CSF findings recovered completely after 3 months later. The first case of West Nile encephalitis in Korea was imported from Guinea, and was cured successfully.

Graphical Abstract

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Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

We developed a real‐time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real‐time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 µl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections. J. Med. Virol. 81:146–152, 2009. © 2008 Wiley‐Liss, Inc.     

West Nile virus encephalitis with myositis and orchitis

This report documents the hospital course and autopsy findings of a 43-year-old man with a renal allograft who died of West Nile virus (WNV) encephalitis. Central nervous system (CNS) findings were those of severe necrotizing and hemorrhagic encephalitis affecting gray matter regions limited to the diencepahlon, rhombencephalon, spinal cord, and limbic system. The bilateral process exhibited preferential involvement of motor neurons. Brain imaging obtained 6 days before death demonstrated an unusual pattern of involvement corresponding with the autopsy findings, confirming that imaging may be a specific diagnostic guide in WNV encephalitis. Extra-CNS findings include myositis with T-lymphocyte infiltration of nerve fibers, suggesting that the virus may reach the CNS via peripheral nerves. Orchitis with dense T-lymphocyte infiltration and syncytial cell formation thought to be due to WNV were also noted.     

Multiple pathways to the attenuation of West Nile virus in South-East Texas in 2003

West Nile virus (WNV) was first detected in Texas in 2002. During 2003, several isolates exhibiting significant attenuation of mouse neuroinvasiveness, and in some cases a small plaque and temperature sensitive phenotype when compared to other North American WNV isolates, were obtained from birds and mosquitoes in South-East Texas. To determine the attenuation markers of WNV, we have sequenced the genomes of three attenuated isolates and four temporally related virulent isolates and compared the amino acid substitutions in a total of 101 isolates, including three previously published genomes of attenuated strains, to identify mutations that are potentially involved in attenuation. Surprisingly, the attenuated strains fall into three separate “groups”, suggesting that the attenuated phenotype evolved on three separate occasions in 2003. None of the groups share the same nucleotide changes or amino acid substitutions, and there are few mutations that can be clearly defined alone as being associated with attenuation.     

Baboon model for West Nile virus infection and vaccine evaluation

Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.     

Characterization of neutralizing antibodies to West Nile virus

We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.     

Seroprevalence of West Nile and dengue virus in the human population of the Bolivian Chaco

To determine the seroprevalence of antibodies against dengue virus (DENV) and West Nile virus (WNV) in the human population of the Bolivian Chaco, we tested 256 inhabitants of two rural communities. The seroprevalence, confirmed by plaque reduction neutralization test, was 7.8% and 2.7% for DENV and WNV, respectively.     

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.     

Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column

Analysis and purification of specific PCR products from PCR reactions can be problematic due to several issues relating to amplification and low product yield. The use of HPLC as a preparative tool in PCR product analysis is common but has not replaced traditional electrophoretic techniques for purifying DNA to be used in subsequent experiments. Gel purification of PCR products can result in a net loss greater than 50% of the starting DNA amount. Thus, this method of recovery can become the limiting factor in the overall cloning protocol. This paper describes a simple and relatively inexpensive micro-preparative HPLC method to purify and analyze nM quantities of DNA. A microbore polyethyleneimine-based anion-exchange column fractionates PCR mixtures in less than 40 min with a recovery of the purified specific product as high as 80%, thus eliminating the need for gel purification. Using this method, nested PCR products from Sindbis virus differing by 18 bp in some cases and a 277 bp fragment from West Nile virus were resolved and quantified. This method differs from existing methodologies because separation is based on size and charge as well as the overall G + C content of the PCR product.     

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。     

Cerebrospinal fluid cytology in seasonal epidemic West Nile virus meningo-encephalitis

The incidence of West Nile Virus (WNV) infection has progressively increased in North America since the first epidemic in 1999. Formal scholarly documentation of cerebrospinal fluid (CSF) cytology changes in patients with WNV infection is limited. We report our experience with CSF cytospins from a population of consecutive patients with documented CSF WNV-specific IgM. Thirty-two patients (12 male, 20 female) with a median age of 52 yr (range, 19-88) diagnosed with WNV meningo-encephalitis were studied. Symptoms were present for a mean of 5 days (range, 1-14) prior to lumbar puncture. CSF proteins were elevated in 94% of patients (30/32) with a mean value of 79 mg/dl (range, 36-185). CSF glucose was normal to elevated in all cases. All cytomorphologically adequate samples demonstrated a pleocytosis with a mean of 156 cells/mm3 (range, 13-683). Nearly, all (26/28) patients showed increased CSF neutrophils--mean 43% (range, 1-83). Mean lymphocyte and monocyte fractions were 44% (range, 8-85) and 14% (range, 2-27), respectively. Three cases showed 1-4% plasma cells. Mean total leukocyte counts (TLC) (197 cells/mm3) and mean neutrophil fractions (50%) were greater in patients sampled within the first 3 days of symptoms than in those sampled beyond day 3 (mean TLC, 126 cells/mm3; mean neutrophil fraction, 37%). Relative lymphocyte proportions increased from a mean of 39 to 48% after 3 days of illness. WNV should be considered as a potential etiology of infectious CSF pleocytosis in the North American late summer and early fall seasons.