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We report 2 cases of febrile lymphocytic meningitis with encephalitis in patients in France. One patient had not traveled outside Paris; the other had traveled to eastern France. Laboratory findings revealed acute West Nile virus infection. The cases occurred days apart, raising concern the virus has spread further in France.

West Nile virus (WNV) lineage 2 is expanding and causing large outbreaks in Europe. In this study, we analyzed the epidemiological, clinical, and virological features of WNV lineage 2 infection during the large outbreak that occurred in northern Italy in 2018. The study population included 86 patients with neuroinvasive disease (WNND), 307 with fever (WNF), and 34 blood donors. Phylogenetic analysis of WNV full genome sequences from patients’ samples showed that the virus belonged to the widespread central/southern European clade of WNV lineage 2 and was circulating in the area at least since 2014. The incidence of WNND and WNF progressively increased with age and was higher in males than in females. Among WNND patients, the case fatality rate was 22%. About 70% of blood donors reported symptoms during follow-up. Within the first week after symptom onset, WNV RNA was detectable in the blood or urine of 80% of patients, while 20% and 40% of WNND and WNF patients, respectively, were WNV IgM-seronegative. In CSF samples of WNND patients, WNV RNA was typically detectable when WNV IgM antibodies were absent. Blunted or no WNV IgM response and high WNV IgG levels were observed in seven patients with previous flavivirus immunity.

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus' low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories' and methods' performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories.

Few reports of human Usutu virus (USUV) infection have been reported to date. We describe the first three patients with USUV neuroinvasive infection in Zagreb and its surroundings from 30 August to 7 September 2013 during a West Nile virus (WNV) outbreak. Patients were aged 29, 56, and 61 years. The two older patients had several comorbidities (arterial hypertension, hyperlipidemia, and diabetes mellitus). All patients presented with meningitis and meningoencephalitis closely resembling WNV neuroinvasive disease. The main clinical features in all patients were headache, fever, nuchal rigidity, hand tremor, and hyperreflexia. Neuroimaging studies were normal and electroencephalography (EEG) revealed diffusely slow activity. The 29 years old, a previously healthy female patient, was deeply somnolent and disoriented for 4 days. Her recovery was slow and even 10 weeks after disease onset, she had memory and speech-fluency difficulties. The other two patients recovered promptly. USUV IgG antibodies were detected in all patients by ELISA with seroconversion documented in two of them. Titers of USUV-neutralizing antibodies were 10, 80, and 10, respectively. Because USUV and WNV share many clinical characteristics, USUV infection could be misdiagnosed as WNV. Testing for USUV should be considered in all suspected cases of meningoencephalitis, especially in areas where both viruses cocirculate.

West Nile virus (WNV) is the most common domestic arbovirus in the United States. During 2018, WNV was transmitted through solid organ transplantation to 2 recipients who had neuroinvasive disease develop. Because of increased illness and death in transplant recipients, organ procurement organizations should consider screening during region-specific WNV transmission months.

To assess the value of multifocal chorioretinitis and of clinical manifestations and biologic parameters in the diagnosis of West Nile virus (WNV) infection. We conducted a prospective, controlled case series study during an outbreak of WNV infection between August 15 and October 24, 2003, of 64 consecutive patients who presented with clinical manifestations consistent with WNV disease. In each patient, standardized clinical and biologic data were collected. An ophthalmologic examination searching particularly for multifocal chorioretinitis was performed. Of 64 patients who presented primarily with meningitis and/or encephalitis, 36 had IgM antibodies against WNV. The WNV-infected patients tended to be older (median age of 54 years vs 46 years in WNV infection and control groups, respectively) and more frequently had diabetes (30% vs 7% in WNV infection and control groups, respectively; P=.03). Multifocal chorioretinitis was found in 75% of WNV-infected patients but in no patient in the control group ( P=.001). Blood glucose and amylase levels were higher in WNV-infected patients, whereas serum sodium levels were lower. The cerebrospinal fluid leukocyte count and protein levels were significantly higher in WNV meningitis or encephalitis. Overall, multifocal chorioretinitis had 100% specificity and 73% sensitivity (88% when only patients with meningitis or encephalitis were analyzed) for the diagnosis of WNV. Multivariate analysis disclosed multifocal chorioretinitis as the only predictor of WNV infection (odds ratio, 62; 95% confidence interval, 6-700; P=.001). Multifocal chorioretinitis appears to be a specific marker of WNV infection, particularly in patients who present with meningoencephalitis. An ophthalmologic examination should be part of the routine evaluation of such patients.

We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.

In Europe, many flaviviruses are endemic (West Nile, Usutu, tick-borne encephalitis viruses) or occasionally imported (dengue, yellow fever viruses). Due to the temporal and geographical co-circulation of flaviviruses in Europe, flavivirus differentiation by diagnostic tests is crucial in the adaptation of surveillance and control efforts. Serological diagnosis of flavivirus infections is complicated by the antigenic similarities among the Flavivirus genus. Indeed, most flavivirus antibodies are directed against the highly immunogenic envelope protein, which contains both flavivirus cross-reactive and virus-specific epitopes. Serological assay results should thus be interpreted with care and confirmed by comparative neutralization tests using a panel of viruses known to circulate in Europe. However, antibody cross-reactivity could be advantageous in efforts to control emerging flaviviruses because it ensures partial cross-protection. In contrast, it might also facilitate subsequent diseases, through a phenomenon called antibody-dependent enhancement mainly described for dengue virus infections. Here, we review the serological methods commonly used in WNV diagnosis and surveillance in Europe. By examining past and current epidemiological situations in different European countries, we present the challenges involved in interpreting flavivirus serological tests and setting up appropriate surveillance programs; we also address the consequences of flavivirus circulation and vaccination for host immunity.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

West Nile virus (WNV) is a neurotropic mosquito-borne orthoflavivirus, representing a relevant public health threat. Identification of biomarkers that would predict the course of WNV infection is of interest for the early identification of patients at risk and for supporting decisions on therapeutic interventions. In this study, serum levels of glial fibrillary acidic protein (sGFAP) and neurofilament light chain (sNfL), which are markers of brain tissue damage and inflammation, were analysed in 103 subjects with laboratory-confirmed WNV infection, comprising 13 asymptomatic blood donors, 23 with WN fever (WNF), 50 with encephalitis/meningoencephalitis (E/ME) and 17 with acute flaccid paralysis (AFP). In addition, 55 WNV-negative subjects with fever, encephalitis or healthy asymptomatic were included as controls. Age-adjusted levels of both sNfL and sGFAP were significantly higher in patients with neuroinvasive disease than in those with fever or asymptomatic (both WNV-positive and WNV-negative), suggesting a broad association of these biomarkers with systemic inflammation and brain injury resulting from infection. In WNV patients, the combined analysis of sNfL and sGFAP early after symptom onset allowed discrimination between neuroinvasive disease and fever with 67.2% sensitivity and 91.3% specificity, but not between E/ME and AFP. Furthermore, high levels of sNfL and sGFAP were significantly associated with prolonged hospital stay, intensive care unit admission and the occurrence of death or severe sequelae. Detection of WNV RNA in CSF was associated with increased sGFAP. In conclusion, our study indicates the potential utility of sNfL and sGFAP as biomarkers of WNV disease severity and adverse outcome.