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Background Recent outbreaks of Zika Virus (ZIKV) infection and associated microcephaly has raised multiple scientific questions. The close antigenic relatedness between flaviviruses makes diagnosis of specific infection difficult. This relatedness also raises the potential of Antibody Dependent Enhancement (ADE) via cross reactive antibodies to flaviviruses like West Nile Virus (WNV) and Dengue Virus (DENV). Asymptomatic WNV infections are endemic throughout the US creating a large proportion of the population that is seropositive for WNV antibodies. Whether these sero-positive individuals potentially carry ZIKV enhancing antibodies remains unknown. Results Serum samples obtained from human subjects with symptomatic or asymptomatic WNV infection from a WNV endemic region in Texas were tested for their ability to enhance or neutralize ZIKV infection. Sero-surveillance data demonstrated a ~ 7% prevalence for WNV antibodies in the population. Sera from both symptomatic and asymptomatic WNV seropositive donors effectively neutralized WNV and to some extent DENV infection. Interestingly, WNV+ sera failed to inhibit ZIKV while significantly enhancing infection. Conversely, ZIKV specific sera effectively neutralized ZIKV, with ADE only evident at lower concentrations. The enhancement of ZIKV via WNV antibody positive sera was likely due to non-neutralizing Envelope (E) antibodies as seen with monoclonal ZIKV E antibodies. Conclusions Overall, our findings suggest that WNV antibodies in the sera significantly enhance ZIKV infection in Fc receptor positive cells with limited neutralization activity. Further studies in more relevant models of ADE will be needed to confirm the relevance of these findings in vivo.

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008–2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.

West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus, humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient&rsquo, s urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.

Immunoglobulin administered to adults with neuroinvasive disease appeared to be safe but was not demonstrated to improve clinical outcomes., West Nile Virus (WNV) can result in clinically severe neurologic disease. There is no treatment for WNV infection, but administration of anti-WNV polyclonal human antibody has demonstrated efficacy in animal models. We compared Omr-IgG-am, an immunoglobulin product with high titers of anti-WNV antibody, with intravenous immunoglobulin (IVIG) and normal saline to assess safety and efficacy in patients with WNV neuroinvasive disease as part of a phase I/II, randomized, double-blind, multicenter study in North America. During 2003–2006, a total of 62 hospitalized patients were randomized to receive Omr-IgG-am, standard IVIG, or normal saline (3:1:1). The primary endpoint was medication safety. Secondary endpoints were morbidity and mortality, measured using 4 standardized assessments of cognitive and functional status. The death rate in the study population was 12.9%. No significant differences were found between groups receiving Omr-IgG-am compared with IVIG or saline for either the safety or efficacy endpoints.

Background Viruses are aetiologic agents of several human infectious diseases, which can be distinguished into acute and chronic. Among the agents causing acute infections, arboviruses, such as Dengue (DENV), West Nile (WNV) and Zika (ZIKV) Viruses, have increasingly spread worldwide. Moreover, the newly discovered Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing the COVID-19 disease, is responsible for an incredibly quantity of infections and deaths. Conversely, the Human Immunodeficiency Virus (HIV-1) is a well-known pathogen able to cause chronic latent infection, but it still represents a global public health concern. The main strategy for combating viral infections is a combination of vaccination and antiviral drugs. However, vaccines are available only for a minority of viral pathogens, thus the demand for new antiviral strategies has significantly increased. Anyway, the screening of candidate compounds requires the assessment of their antiviral effects in vitro. Results and discussion An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV, able to distinguish between the inhibitory effect of molecules targeting the early and post-budding phases of viral replication cycle. Considering that the activity of sofosbuvir, a nucleotide analog licensed for HCV infection, has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. We determined for the first time the antiviral activity of sofosbuvir against WNV in both cell-based and enzymatic assays. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern. The ?ORIGINALE CHEMIAE in Antiviral Strategy? project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Antiviral activity of molecules was determined in vitro against DENV, WNV, HIV-1 and SARS-CoV-2. Two compounds were able to inhibit viral replication against two different families of viruses (DENV/WNV and SARS-CoV-2), and two compounds were able to inhibit viral replication against viruses with remarkable differences in their replication cycle (DENV/WNV and HIV-1). At the beginning of the pandemic, an Italian network named SCIRE (SARS-CoV-2 Italian Research Enterprise) was created in order to trace SARS-COV-2 evolution. By NGS whole genome sequencing, it was defined that the initial outbreak in Italy was mainly attributable to the SARS-CoV-2 lineage B.1 and to its uncontrolled circulation for an estimated period of 4 weeks. Developing strategies to eradicate chronic HIV-1 infection are a high priority. Recently, it was hypothesized that maraviroc (MVC), may exert a latency reversing effect in addition to its antiviral activity. Thus, the MVC-mediated HIV-1 induction was explored. An increased HIV-1 expression was detected at the highest MVC concentration in two of the three cell line models tested. In ex vivo CD4 T cells, MVC-mediated induction was detected sparsely on individual samples. Such evidences suggest the role of MVC as a weak latency reversing agent of the HIV-1 provirus induction. Conclusions Control of viral infections is a continuous challenge for public health. The development of effective antiviral compounds could be reasonably the key to halt the spread or to mitigate severe clinical manifestations of acute viral diseases. Moreover, antiviral therapy is essential to the maintenance of undetectable viral loads in chronic infection. Furthermore, developing of compounds able to exert a reversal latency effect may be a remarkable benefit in exacerbate chronic viral infections. In conclusion, the key concept of this thesis is to underline the strong need, and partially participate, in enhancing antiviral therapies strategies to counteract the burden of both acute and chronic viral diseases.

Background Climate is a major factor in the epidemiology of West Nile virus (WNV), a pathogen increasingly pervasive worldwide. Cases increased during 2018 in Israel, the United States and Europe. Aim We set to retrospectively understand the spatial and temporal determinants of WNV transmission in Israel, as a case study for the possible effects of climate on virus spread. Methods We employed a suitability index to WNV, parameterising it with prior knowledge pertaining to a bird reservoir and Culex species, using local time series of temperature and humidity as inputs. The predicted suitability index was compared with confirmed WNV cases in Israel (2016–2018). Results The suitability index was highly associated with WNV cases in Israel, with correlation coefficients of 0.91 (p value = 4 × 10− 5), 0.68 (p = 0.016) and 0.9 (p = 2 × 10− 4) in 2016, 2017 and 2018, respectively. The fluctuations in the number of WNV cases between the years were explained by higher area under the index curve. A new WNV seasonal mode was identified in the south-east of Israel, along the Great Rift Valley, characterised by two yearly peaks (spring and autumn), distinct from the already known single summer peak in the rest of Israel. Conclusions By producing a detailed geotemporal estimate of transmission potential and its determinants in Israel, our study promotes a better understanding of WNV epidemiology and has the potential to inform future public health responses. The proposed approach further provides opportunities for retrospective and prospective mechanistic modelling of WNV epidemiology and its associated climatic drivers.

West Nile virus (WNV) is a mosquito-borne virus of public health importance. Currently, there is no FDA approved vaccine available against WNV infection in humans. Therefore, the early diagnosis of the WNV infection is important for epidemiologic control and timely clinical management in areas where multiple Flaviviruses are endemic. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that rapidly and accurately detects the envelope (env) gene of WNV. RT-PSR assay was optimized at 63°C for 60 min using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The standard curve for RT-PSR assay was generated using the 10-fold serial dilutions of in vitro transcribed WNV RNA. To determine the detection limit of RT-PSR assay, an amplified product of conventional RT-PCR was in vitro transcribed as per standard protocol. The detection limit of the newly developed RT-PSR assay was compared with that of conventional RT-PCR and CDC reported TaqMan real-time RT-PCR using a serial 10-fold dilution of IVT WNV RNA. The detection limit of RT-PSR was found to be 1 RNA copy, which is 100-fold higher than that of conventional RT-PCR (100 copies). This suggests that RT-PSR assay is a valuable diagnostic tool for rapid and real-time detection of WNV in acute-phase serum samples. The assay was validated with a panel of 107 WNV suspected human clinical samples with signs of acute posterior uveitis and onset of febrile illness. Out of 107 samples, 30 were found positive by RT-PSR assay. The specificities of the selected primer sets were established by the absence of cross-reactivity with other closely related members viruses of the Flaviviruses, Alphaviruses, and Morbilliviruses groups. No cross-reactivity was observed with other viruses. To best of our knowledge, this is the first report describing the RT-PSR assay for the detection of RNA virus (WNV) in clinical samples. RT-PSR is a high throughput method and more than 30 reactions can be run at once in real-time turbidimeter. PSR assay has potential to be used for a rapid screening of large number of clinical samples in endemic areas during an outbreak.

International audience; West Nile virus (WNV) is one of the most widely distributed mosquito-borne viruses in the world. In North Africa, it causes human cases of meningoencephalitis with fatalities in Algeria and in Tunisia, whereas only horses were affected in Morocco. The aims of this study were to detect WNV in mosquitoes and to determine seroprevalence of WNV in Moroccan horses by the detection of IgG antibodies. A total of 1455 mosquitoes belonging to four different species were grouped by collection site, date, and sex with 10 specimens per pool and tested for 38 arboviruses using a high-throughput chip based on the BioMark Dynamic array system. Out of 146 mosquito pools tested, one pool was positive for WNV. This positive pool was confirmed by real time RT-PCR. The serosurvey showed that 33.7% (31/92) of horses were positive for competitive enzyme-linked im-munosorbent assay (cELISA) test. The flavivirus-sphere microsphere immnoassay (MIA) test, targeting three flaviviruses (WNV, Usutu virus (USUV) and Tick borne encephalitic virus (TBEV)) showed that 23 sera out of 31 were positive for WNV, two for USUV, two for USUV or WNV, and four for an undetermined flavivirus. Virus neutralization tests with USUV and WNV showed that 28 of 31 sera were positive for WNV and all sera were negative for USUV. This study reports, for the first time, the detection of WNV from Culex pipiens mosquitoes in Morocco and its circulation among horses. This highlights that the detection of arboviruses in mosquitoes could serve as an early warning signal of a viral activity to prevent future outbreaks in animals and humans.

The economic burden of West Nile virus (WNV) infection is not known for Canada. We sought to describe the direct and indirect costs of WNV infection in the province of Quebec, Canada, up to 2 years after onset of signs and symptoms. We conducted a retrospective cohort study that included WNV cases reported during 2012 and 2013. For 90 persons infected with WNV, persons with encephalitis accounted for the largest proportion of total cost: a median cost of $21,332 per patient compared with $8,124 for West Nile meningitis (p = 0.0004) and $192 for West Nile fever (p

For patients with meningitis and encephalitis, testing for these viruses is underutilized., In the United States, the most commonly diagnosed arboviral disease is West Nile virus (WNV) infection. Diagnosis is made by detecting WNV IgG or viral genomic sequences in serum or cerebrospinal fluid. To determine frequency of this testing in WNV-endemic areas, we examined the proportion of tests ordered for patients with meningitis and encephalitis at 9 hospitals in Houston, Texas, USA. We identified 751 patients (567 adults, 184 children), among whom 390 (52%) experienced illness onset during WNV season (June–October). WNV testing was ordered for 281 (37%) of the 751; results indicated acute infection for 32 (11%). Characteristics associated with WNV testing were acute focal neurologic deficits; older age; magnetic resonance imaging; empirically prescribed antiviral therapy; worse clinical outcomes: and concomitant testing for mycobacterial, fungal, or other viral infections. Testing for WNV is underutilized, and testing of patients with more severe disease raises the possibility of diagnostic bias in epidemiologic studies.

Introduction: We present here a case of West Nile Virus (WNV) encephalitis that initially presented with diabetic ketoacidosis and rhabdomyolysis. Case presentation: A 35-year-old male with no past medical history presented to the emergency department complaining of polydipsia, generalized weakness, lightheadedness, and visual disturbances of one week duration. He was found to be in diabetic ketoacidosis. His hemoglobin A1c was 11%. The patient was appropriately treated for diabetic ketoacidosis and it resolved on hospital day 1. On hospital day 2, the patient developed a fever of 101.6 °F and his mental status became severely altered. He developed auditory and visual hallucinations. IgM and IgG antibodies to West Nile Virus were positive in the cerebral spinal fluid (CSF). The patient's creatine kinase level rose to 118,400 U/L during his hospitalization and eventually returned to baseline. The patient made a full recovery with no residual neurologic deficits after an 11 day hospital course. Discussion: In this patient, neuroinvasive WNV was confirmed with positive CSF IgM. The patient’s newly diagnosed diabetes likely contributed to his susceptibility to neuroinvasive disease. Furthermore, WNV encephalitis in a background of DKA has not been previously described in the literature and this case demonstrates WNV neuroinvasive disease should be in the differential diagnosis for patients presenting with unexplained neurological symptoms. Conclusion: Diagnosis of neuroinvasive WNV is imperative to stop unnecessary therapies, limit further diagnostic evaluation, help predict patient outcomes, direct public health prevention measures, and further provide investigations into the clinical conditions that define the spectrum of WNV disease. Keywords: DKA, Diabetic ketoacidosis, West Nile Virus, WNV, Rhabdomyolysis, Neuroinvasive

West Nile Virus (WNV) is an arthropod-borne flavivirus whose zoonotic cycle includes both mosquitoes and birds as amplifiers and humans and horses as dead-end hosts. In recent years WNV has been spreading globally and is currently endemic in Africa, The Middle East, India, Australia, central and southern Europe, and the Americas. Integrated surveillance schemes and environmental data aim to detect viral circulation and reduce the risk of infection for the human population emphasizing the critical role for One Health principles in public health. Approximately 20% of WNV infected patients develop West Nile Fever while in less than 1%, infection results in West Nile Neurological Disease. Currently, the diagnosis of WNV infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia. The recent emergence of Zika virus epidemic in America and Asia has added another layer of complexity to WNV diagnosis due to significant cross-reactivity between several members of the Flaviviridae family such as Zika, dengue, Usutu, and West Nile viruses. Diagnosis is especially challenging in persons living in regions with flavivirus co-circulation as well as in travelers from WNV endemic countries traveling to Zika or dengue infected areas or vise-versa. Here, we review the recent studies implementing WNV surveillance of mosquitoes and birds within the One Health initiative. Furthermore, we discuss the utility of novel molecular methods, alongside traditional molecular and serological methods, in WNV diagnosis and epidemiological research.

Given the pivotal role of monocyte-derived dendritic cells (DCs) in determining the magnitude of the antiviral innate immune response, we sought to determine whether Usutu virus (USUV) and West Nile virus (WNV) lineages (L)1 and L2 can infect DCs and affect the rate of type I interferon (IFN) activation. The sensitivity of these viruses to types I and III IFNs was also compared. We found that USUV can infect DCs, induce higher antiviral activities, IFN alpha subtypes and the IFN stimulated gene (ISG)15 pathway, and is more sensitive to types I and III IFNs than WNVs. In contrast, we confirmed that IFN alpha/beta subtypes were more effective against WNV L2 than WNV L1. However, the replication kinetics, induction of IFN alpha subtypes and ISGs in DCs and the sensitivity to IFN lambda 1–3 did not differ between WNV L1 and L2.

A mutation leading to substitution of a key amino acid in the prM protein of West Nile virus (WNV) occurred during persistent infection of an immunocompetent patient. WNV RNA persisted in the patient’s urine and serum in the presence of low-level neutralizing antibodies. This case demonstrates active replication of WNV during persistent infection.

We sequenced West Nile viruses (WNVs) from Turkey and found close relationships to WNV lineage 1 strain ArB310/67 from the Central African Republic, distinct from other WNVs circulating in the Mediterranean Basin, eastern Europe, and the Middle East. These findings suggest independent introductions of WNV strains from Africa to the Middle East.

These birds are highly susceptible to strains circulating in Europe and, thus, may serve as surveillance sentinels., West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains’ virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

West Nile virus (WNV), the most widespread flavivirus, is distributed throughout Africa, Asia, Europe, and Australia, and since 1999, WNV has also been present in the Americas (1). Within the last 2 decades, WNV infection has caused an increasing number of cases of neuroinvasive disease in humans and become a substantial public health problem (1). Up to 8 lineages of WNV, based on genetic differences, have been proposed (1,2) (Table 1). Lineage 1 is widely distributed and further divided into lineage 1a, which includes the American strains; lineage 1b, which is also referred to as Kunjin virus and mainly described in Australia; and lineage 1c, which is also referred to as lineage 5 and comprises isolates from India. Lineage 2 has been detected in Africa and several parts of Europe, lineage 3 (Rabensburg virus) has been isolated only in the Czech Republic, and lineage 4 has been reported from Russia (3). A putative sixth lineage, based on a small genome fragment, has been described from Spain (4), and putative lineages 7 (Koutango virus) and 8 have been reported from Senegal (2). Table 1 Overview of West Nile virus lineages WNV is maintained in an enzootic cycle between mosquitoes and wild birds (1). In 2013, ≈100 Uranotaenia unguiculata Edwards, 1913, mosquitoes were trapped during mosquito-monitoring projects at Lake Neusiedl-Seewinkel National Park in Austria and near Sedlec in the Czech Republic. In Russia, Ur. unguiculata mosquitoes have been described as hosting lineage 4 WNV strains (A. Platonov, unpub. data) (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"FJ154906","term_id":"204324973","term_text":"FJ154906"}}FJ154906–49 and {"type":"entrez-nucleotide","attrs":{"text":"FJ159129","term_id":"204305343","term_text":"FJ159129"}}FJ159129–31). To determine whether Ur. unguiculata mosquitoes in Austria and the Czech Republic also host WNV, we investigated the mosquitoes collected in 2013 for the presence of WNV, focusing on lineage 4 viruses.

West Nile virus (WNV) is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3’ untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid (C)/Capsid Anchor (CA) junction of the viral polyprotein. Recombinant viruses containing a TAT(1-67) or Gaussia Luciferase (GLuc) gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C/CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints.

To establish the presence of West Nile virus (WNV) infection in the animal population in Serbia after the human WNV outbreak, the presence of anti-WNV IgG antibodies was examined by commercial ELISA of blood sera samples of 130 horses collected in 2012 from 6 stables and 1 settlement in Vojvodina Province, northern Serbia. During the blood sampling, hibernating mosquitoes in the vicinity of the sampled horses were collected (31 pools from 4 locations) and tested for WNV presence by real-time RT-PCR. The presence of anti-WNV antibodies was observed in 49.23% (64/130) horses. Per stable, the percent of seropositive animals ranged from 35% to 64%. All 31 analyzed pools of hibernating mosquitoes tested negative for WNV RNA. The WNV-antibody prevalence of 49.23% obtained in horses during 2012 was much higher than the prevalence (12%) found in horses during 2009/2010. These results, including the confirmed seroconversion in eight horses that tested negative in 2010, indicated an intensive WNV circulation during 2012 in Serbia, and the necessity of implementing surveillance programs. [Projekat Ministarstva nauke Republike Srbije, br. TR31084 and III43007]

We investigated the genetics and evolution of West Nile virus (WNV) since initial detection in the United States in 1999 on the basis of continual surveillance studies in the Houston, Texas, USA, metropolitan area (Harris County) as a surrogate model for WNV evolution on a national scale. Full-length genomic sequencing of 14 novel 2010–2012 WNV isolates collected from resident birds in Harris County demonstrates emergence of 4 independent genetic groups distinct from historical strains circulating in the greater Houston region since 2002. Phylogenetic and geospatial analyses of the 2012 WNV isolates indicate closer genetic relationship with 2003–2006 Harris County isolates than more recent 2007–2011 isolates. Inferred monophyletic relationships of these groups with several 2006–2009 northeastern US isolates supports potential introduction of a novel WNV strain in Texas since 2010. These results emphasize the need to maintain WNV surveillance activities to better understand WNV transmission dynamics in the United States.

Ozan, Emre/0000-0001-9872-8152 WOS: 000318926100011 PubMed: 23785699 Background: West Nile virus (WNV) and Rift Valley fever virus (RVFV) are mosquito-borne viral diseases. The objective of this study was to investigate the RVFV and WNV infections as serologically in different mammalian species (cattle, horse, goat, sheep and water buffalo) in the northern Turkey. Methods: Blood samples randomly collected from 70 each cattle, horse, sheep, goat and water buffalo were analyzed for the presence of antibodies to RVFV and WNV using an competitive enzyme-linked immunosorbent assay (C-ELISA) in northern Turkey. Results: None of the animals were positive for antibodies to RVFV. In contrast, WNV antibodies were found in two of 350 samples (0.57%). Conclusion: This may suggest that the RVFV disease is not present in northern Turkey. This is the first serological study on RVFV in Turkey.

Background To date there has been no evidence of mosquito-borne virus transmission of public health concern in the UK, despite the occurrence of more than 30 species of mosquito, including putative vectors of arboviruses. The saltmarsh mosquito Ochlerotatus detritus [syn. Aedes (Ochlerotatus) detritus] is locally common in parts of the UK where it can be a voracious feeder on people. Methods Here, we assess the competence of O. detritus for three major arboviruses: dengue virus (DENV), chikungunya virus (CHIKV) and West Nile virus (WNV) using adult mosquitoes reared from wild, field-obtained immatures. Results We demonstrate laboratory competence for WNV at 21 °C, with viral RNA detected in the mosquito’s saliva 17 days after oral inoculation. By contrast, there was no evidence of laboratory competence of O. detritus for either DENV or CHIKV. Conclusions To our knowledge, this is the first study to demonstrate competence of a UK mosquito for WNV and confirms that O. detritus may present a potential risk for arbovirus transmission in the UK and that further investigation of its vector role in the wild is required.

We investigated West Nile virus (WNV) as a possible disease etiology in persons hospitalized in South Africa. Of 206 specimens tested, 36 had WNV neutralizing antibodies, significantly more than in similar earlier serosurveys. Seven probable acute WNV cases were identified, suggesting WNV may be overlooked as an etiology of severe disease in South Africa.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) present ecological and antigenic similarities and are responsible for serious human diseases. In addition, WNV is a significant pathogen in terms of equine health. The purpose of our study was to analyse the seroprevalence of SLEV and WNV in equine sera collected in Santa Fe Province, Argentina. The seroprevalence determined using the plaque reduction neutralisation test was 12.2% for SLEV, 16.2% for WNV and 48.6% for a combination of both viruses. These results provide evidence of the co-circulation of SLEV and WNV in equines in Santa Fe.

West Nile virus (WNV) envelope glycoproteins preM/E were stably expressed in baby hamster kidney cells and tested as antigen in a fluorescent antibody assay for WNV antibodies. Sera from horses, mice and chicken immunized with an inactivated WNV vaccine and, less consistently, sera from horses acutely infected with WNV, reacted specifically with viral antigens present in preM/E-expressing cells.

To the Editor: West Nile fever is caused by the West Nile virus (WNV), a mosquito-borne member of the genus Flavivirus. Birds are the natural reservoir of the virus, which is maintained in nature in a mosquito–bird–mosquito transmission cycle. WNV has been detected in many regions worldwide, including North America, Europe, Africa, the Near East, and Asia (1). WNV has been shown to cause meningoencephalitis in humans and horses. In the United States, seroconversion in dogs was detected 6 weeks before a human case was reported (2). Thus, dogs could be considered as sentinels for WNV infection, but their role as reservoir is unlikely because of short-term and low levels of viremia (3). In this study, we determined the seroprevalence of WNV in dogs living close to humans in different environments to assess their role as sentinels of this potentially severe zoonosis. During 2003–2012, blood samples were collected from 753 adult dogs from France and 6 countries in Africa (Table). Samples were centrifuged within 24 h after collection, separated, frozen at –20°C, and sent to the virology laboratory of the Institut de Recherche Biomedicale des Armees (Marseille, France). Each sample was systematically tested for IgG against WNV by using an in-house ELISA with inactivated WNV as antigen. Serum samples were considered positive if the optical density at 450 nm was >3-fold the mean of that for negative antigen. Because of the antigenic cross-reactivity among flaviviruses, all positive samples were further tested by Western blot for WNV-specific antibodies (4); seroprevalence was calculated on the basis of Western blot–confirmed cases only. For the statistical analysis, we used the exact binomial method to calculate 95% CIs of the proportions and the Fisher exact test to calculate p values and compare the seroprevalence rates between countries; significance was set at p

Age-related macular degeneration (AMD) is the leading cause of blindness in the developing world in people aged over 60 years, manifested as a loss of central vision in one or both eyes, with significant morbidity including loss of mobility and depression. This condition involves the degeneration of the macula, and although the exact aetiology of this disease is unknown, various epidemiological studies have shown it to be multifactorial. Current research points towards the involvement of a dysregulated immune system in the pathogenesis and progression of the disease: as the body ages, the immune system increasingly adopts a more inflammatory basal state. However, not all of the aged population develops AMD and it is highly likely that an additional stimulus or stimuli is/are needed to exploit this dysregulated immune environment to initiate this disease. Given the range of pathogens that can infect the retina, we hypothesize that this breaking point could manifest as a chronic inflammation as a result of a low-level infection. West Nile Virus (WNV) is a flavivirus that has come into international prominence ever since its spread into previously WNV-free regions following the 1999 New York outbreak. As several case reports have shown that WNV is capable of infecting the retina, and given its immunopathogenic properties, we believe the virus is a useful tool to model key immune pathways and responses that may be involved in the development and progression of AMD. Of significant interest are the processes involved in the breakdown of the outer blood-retinal barrier (BRB), which is an important step in the progression of AMD from an early stage to a more severe one. Additionally, deciphering and understanding the profile and populations of leukocytes that are recruited during an immunopathic infection in an organ regarded as being immunoprivileged is of great appeal. With this in mind, we set out to investigate the effects of WNV infection on the retinal pigment epithelium (RPE), which comprises the outer BRB. Previously, our laboratory established the WNV BRB model by quantitating various parameters, such as level of infectivity, viral output by WNV-infected RPE and effects of WNV infection on RPE proliferation/migration. The effect of WNV on the extracellular matrix (ECM) production by RPE was also investigated and increases in collagen I, IV and fibronectin were noted. Global ECM production induced a lowered rate of proliferation of RPE seeded on WNV-infected RPE ECM as opposed to mock-infected ECM. A full genome microarray was also undertaken on WNV-infected RPE to analyse differentially regulated gene mRNA production, and increases in several immune genes, as well as genes involved in the stress-response pathway and the TGFβ pathway were found. This current investigation expanded upon these results, and found that WNV infection produces a predominantly CCL5 chemokine response rather than a CCL2 response. Additionally, a lack of TNF production was noted, despite a high initial upregulation of the TNF gene in WNV-infected cells. WNV attenuation was found to be predominantly IFNβ-1-driven, while induction of indoleamine 2,3 dioxygenase activity was induced in part by IFNλ-1 and -2. The effects of WNV infection on RPE barrier integrity was investigated, and an initial increase in infected cells of barrier integrity was observed. Several investigations resulted in a conclusion of a soluble-mediator as the likely mechanism behind this initial increase, and while none of the chemokines tested appeared to contribute to this change, the results suggest that it may be TLR3/RIG-I independent. Finally, establishment of a murine WNV intravitreal model was also undertaken, and several key parameters were determined, including confirmation of WNV-infection of the murine retina, effect of WNV titre on mortality, and histological analysis of the effects of WNV infection on the murine retina. Quantification of the leukocyte profile recruited into the WNV-infected murine retina and choroid revealed significant increases in inflammatory Ly6Chi monocytes, as well as significant differences between immune mice and naïve mice intravitreally infected with WNV, and differences between 2 month old and 5 month old mice. Collectively, these results highlight the importance of the interferon response in both direct and indirect anti-WNV activities and immunomodulation, the changes in outer BRB integrity and possible contributors to its degradation, and the establishment of the murine intravitreal WNV model along with identification of several key leukocytes that are recruited at the peak of infection. These results will help guide further research and highlight possible immune pathways that may contribute to dysregulated inflammatory processes that may occur during the pathogenesis of AMD.

International audience; OBJECTIVE: To assess the value of multifocal chorioretinitis and of clinical manifestations and biologic parameters in the diagnosis of West Nile virus (WNV) infection.PATIENTS AND METHODS: 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.RESULTS: 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).CONCLUSION: 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.

© 2015 Elsevier Inc. Flaviviruses are RNA viruses that constitute a worrisome threat to global human and animal health. In Europe, West Nile virus (WNV) outbreaks have dramatically increased in number and severity in recent years, with dozens of human and horse deaths and a high avian mortality across the continent. Besides WNV, the only clinically relevant mosquito-borne flavivirus detected so far in Europe has been the Usutu virus (USUV), which after being reported for the first time in Austria in 2001, quickly spread across Europe, causing a considerable number of bird deaths and neurological disorders in a few immunocompromised patients. Even though USUV infects multiple avian species that develop antibodies, there is little information about USUV susceptibility, pathogenicity and cross-reactive immunity. Here, the susceptibility of suckling and adult mice to USUV infection and the induction of cross-protective immunity against WNV challenge have been addressed., Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), PLATESA (P2013/ABI-2906) from the Comunidad Autónoma de Madrid, and by the Network of Animal Disease Infectiology and Research-European Union (NADIR-EU- 228394)

Specimens from a patient of the 2000 Israel West Nile virus epidemic were analyzed by reverse transcription-polymerase chain reaction. Products corresponding to E, NS3, and NS5 sequences were amplified from cerebellar but not from cortical samples. Phylogenetic analyses indicated a closer relationship of this isolate to 1996 Romanian and 1999 Russian than to 1998-99 Israeli or 1999 New York isolates.

The viral determinants governing the varied neuropathogenicity of different West Nile virus (WNV) strains are poorly understood. Here, we generated an infectious clone (WNV-MAD(IC)) of the non-pathogenic strain WNV-MAD78 and compared its replication to that of parental WNV-MAD78 and a WNV-MAD78 infectious clone (WNV-MAD(TX-UTRs)) containing the 5' and 3' untranslated regions (UTRs) of the pathogenic strain WNV-TX. All three viruses replicated at similar rates and caused similar lethality in mice. Thus, the infectious clone is indistinguishable from parental virus in replication and neurovirulence, and the UTRs alone do not account for the increased virulence of WNV-TX compared to WNV-MAD78.

To control West Nile virus (WNV), it is necessary to know which mosquitoes are able to transmit this virus. Therefore, we evaluated the WNV vector potential of several North American mosquito species. Culex restuans and Cx. salinarius, two species from which WNV was isolated in New York in 2000, were efficient laboratory vectors. Cx. quinquefasciatus and Cx. nigripalpus from Florida were competent but only moderately efficient vectors. Coquillettidia perturbans was an inefficient laboratory vector. As WNV extends its range, exposure of additional mosquito species may alter its epidemiology.

West Nile virus, an Old World flavivirus related to St. Louis encephalitis virus, was first recorded in the New World during August 1999 in the borough of Queens, New York City. Through October 1999, 62 patients, 7 of whom died, had confirmed infections with the virus. Ornithophilic mosquitoes are the principal vectors of West Nile virus in the Old World, and birds of several species, chiefly migrants, appear to be the major introductory or amplifying hosts. If transovarial transmission or survival in overwintering mosquitoes were the principal means for its persistence, West Nile virus might not become established in the New World because of aggressive mosquito suppression campaigns conducted in the New York area. However, the pattern of outbreaks in southern Europe suggests that viremic migratory birds may also contribute to movement of the virus. If so, West Nile virus has the potential to cause outbreaks throughout both temperate and tropical regions of the Western Hemisphere.

West Nile Virus (WNV) is a mosquito-borne virus and the leading cause of arboviral (arthropod-borne) disease in the U.S. While most WNV cases are asymptomatic, 20% of infected people develop WNV fever and < 1% develop severe neurologic disease. Individuals over 50 years old are at greatest risk of severe disease and death. Dramatic increases in WNV activity in 2012 underscored its unpredictable nature and highlighted concerns for adverse effects on older adults. It is important to understand factors that influence this population's engagement in WNV prevention. This study analyzed data collected by the Maryland Department of Health and Mental Hygiene (DHMH) via cross-sectional survey to identify barriers to WNV prevention among adults ≥60 years of age. Subjects were recruited via stratified random sample of 1,700 households from counties with ≥ two WNV cases, enrolling 211 Maryland adults ≥ 60 years old. Six constructs of the Health Belief Model (HBM)--perceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action, and self-efficacy--were examined to assess how they predicted attitudes and behaviors toward WNV prevention. Univariate, bivariate and multivariate analyses examined the utility of the HBM for explaining WNV preventive behaviors in Maryland adults. Multivariate logistic regression models tested 36 hypotheses examining associations between HBM constructs and six outcomes: (1) avoiding the outdoors at dusk and dawn, (2) dressing in long-sleeved shirts and long pants when outdoors, (3) using insect repellent on exposed skin, (4) draining standing water from objects around one's property, (5) acceptance of a WNV vaccine, and (6) support for community mosquito control programs. Findings showed high WNV knowledge and awareness but low perceptions of personal risk for WNV infection. Perceived susceptibility to WNV predicted use of insect repellent, draining of standing water from objects around the home, and acceptance of a WNV vaccine; perceived benefits were associated with draining standing water and support for mosquito control programs. Feelings of worry about WNV may inform future WNV interventions and risk communication to older adults. Findings have implications for theory-based research, which could probe applications of the HBM and other theories in understanding WNV attitudes and behaviors.

Following its spread in the USA, West Nile Virus (WNV) has reemerged in the Mediterranean basin with a renewed pathogenicity. The introduction of WNV lineage 2 in Europe and its co-circulation with lineage 1 has resulted in a continuously changing epidemiological scenario, highlighting the importance of differential detection of the two lineages. The paper describes a new real-time PCR method for the detection and genotyping of the two main lineages of WNV. The method requires a single pair of primers and probes and is based on the analysis of highly conserved consensus sequences detected in the 5′ terminus of the viral genome.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies.

Background Enveloped viruses utilize cellular membranes to bud from infected cells. The process of virion assembly and budding is often facilitated by the presence of certain conserved motifs within viral proteins in conjunction with cellular factors. We hence examined the West Nile Virus (WNV) Envelope protein for the presence of any such motifs and their functional characterization. Results We identified conserved 461PXAP464 and 349YCYL352 motifs in the WNV envelope glycoprotein bearing resemblance to retroviral late domains. Disruptive mutations of PXAP to LAAL and of the highly conserved Cys350 in the YCYL motif, led to a severe reduction in WNV particle production. Similar motifs in case of retroviruses are known to interact with components of host sorting machinery like PXAP with Tsg101 and YXXL with Alix. However, in the case of WNV, siRNA mediated depletion of Alix or Tsg101 did not have an effect on WNV release. Molecular modeling suggested that while the 461PXAP464 motif is surface accessible and could potentially interact with cellular proteins required for WNV assembly, the 349YCYL352 motif was found to be internal with Cys350 important for protein folding via disulphide bonding. Conclusions The conserved 461PXAP464 and 349YCYL352 motifs in the WNV envelope are indispensable for WNV particle production. Although these motifs bear sequence similarity to retroviral late domains and are essential for WNV assembly, they are functionally distinct suggesting that they are not the typical late domain like motifs of retroviruses and may play a role other than Alix/Tsg101 utilization/dependence.

We identified West Nile virus (WNV) RNA in skin, fat, muscle, tendon, and bone marrow from a deceased donor associated with WNV transmission through solid organ transplantation. WNV could not be cultured from the RNA-positive tissues. Further studies are needed to determine if WNV can be transmitted from postmortem tissues.

The development and persistence of anti-West Nile Virus (WNV) immunoglobulin G (IgG)- and IgM-specific antibodies were investigated in 68 asymptomatic blood donors (BDs) previously tested as positive between October, 2008, and September, 2009, and living in northeastern Italy. Our study showed that WNV-specific IgG titers became negative (41%) or decreased (33%) in a large percentage of BDs, while they increased in a smaller percentage (10%); 16% of BDs showed no titer variation. Reversion to seronegative status within a short time frame suggests that WNV surveillance should be maintained year after year.

The objective of this study was to identify the impact of West Nile virus (WNV) and Toscana virus (TOSV) in febrile diseases of unknown aetiology in Eastern Thrace, Turkey; this study was conducted during August-October 2012, and included 18 clinical cases and 296 blood donors for local serosurveillance. Antibodies were determined via commercial assays and further tested for specificity via neutralization assays (NA). Viral RNAs were sought via specific and/or generic primers. WNV infections were diagnosed in seven patients (38.8%), detected via RNA+IgM in four, RNA in one and IgM and low avidity IgG in two cases. The most common symptom was fever (>38 degrees C), followed by headache, malaise/fatigue, myalgia/arthralgia, muscle stiffness/lower back pain, anorexia, nausea/vomiting, diarrhoea, supraorbital/retrobulbar pain and abdominal pain. Neurological symptoms were noted in one individual. WNV strains in RNA-detectable patients were characterized as lineage 1. TOSV RNA or IgM were identified in two individuals with confirmed WNV infections and in one patient without evidence of WNV exposure. The clinical and laboratory findings in individuals with WNV/TOSV co-infection were comparable to those in WNV-induced disease. The TOSV strain in the patient with detectable viral RNA was characterized as genotype A. In local blood donors, seroreactivity for specific WNV and TOSV immunoglobulins was observed in 1.7% (5/296) and 14.4% (26/180), respectively. These findings indicate the emergence of WNV and TOSV-associated diseases in Eastern Thrace. WNV/TOSV co-infections were documented for the first time.

Summary Background West Nile fever is an important zoonotic infection caused by West Nile virus (WNV), a member of the Flaviviridae . Previous serological data from Turkey suggest widespread WNV circulation. This report includes cases of human and equine WNV infections occurring concurrently, and manifesting as central nervous system infections, in two neighboring provinces of Central Anatolia, Turkey. A partial phylogenetic analysis of the causative virus is given for the first time. Methods The cases were reported in February (horses) and March (human). Symptoms of the disease were similar in the two species, characterized by neurological manifestations suggesting meningoencephalitis. Real-time/nested PCRs and commercial immunoassays and a plaque reduction neutralization assay were employed for the detection of viral RNA and specific antibodies, respectively. Results WNV RNAs were detected in buffy coat (horses) and cerebrospinal fluid (human) samples. Partial nucleotide sequences of the E-gene coding region revealed that the strains are closely related to viruses of lineage 1, clade 1a. Accompanying equine serosurveillance demonstrated WNV-specific antibodies in 31.6% of the samples. Conclusions This is the first report of acute WNV infections caused by lineage 1 strains from Turkey, in concordance with previous reports from some European and North African countries.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:45:39Z No. of bitstreams: 1 S0074-02762012000800020.pdf: 277444 bytes, checksum: e8adbafbaf6ed7a1f63e4a9f9131fe9f (MD5) Made available in DSpace on 2013-08-22T18:45:39Z (GMT). No. of bitstreams: 1 S0074-02762012000800020.pdf: 277444 bytes, checksum: e8adbafbaf6ed7a1f63e4a9f9131fe9f (MD5) Previous issue date: 2012-12-01 Made available in DSpace on 2013-09-30T18:07:34Z (GMT). No. of bitstreams: 2 S0074-02762012000800020.pdf: 277444 bytes, checksum: e8adbafbaf6ed7a1f63e4a9f9131fe9f (MD5) S0074-02762012000800020.pdf.txt: 10803 bytes, checksum: 623184370d2a5b97c8097b5a5ee72b69 (MD5) Previous issue date: 2012-12-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:25:22Z No. of bitstreams: 2 S0074-02762012000800020.pdf: 277444 bytes, checksum: e8adbafbaf6ed7a1f63e4a9f9131fe9f (MD5) S0074-02762012000800020.pdf.txt: 10803 bytes, checksum: 623184370d2a5b97c8097b5a5ee72b69 (MD5) Made available in DSpace on 2014-05-20T13:25:22Z (GMT). No. of bitstreams: 2 S0074-02762012000800020.pdf: 277444 bytes, checksum: e8adbafbaf6ed7a1f63e4a9f9131fe9f (MD5) S0074-02762012000800020.pdf.txt: 10803 bytes, checksum: 623184370d2a5b97c8097b5a5ee72b69 (MD5) Previous issue date: 2012-12-01 In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal. Fiocruz Instituto Oswaldo Cruz Laboratório de Diptera Centers for Disease Control and Prevention Faculdade de Medicina de São José do Rio Preto (FAMERP) Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Mount Sinai School of Medicine Massachusetts Institute of Technology Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas

Background West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals. Results We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed −1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. Conclusions N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

The aim of this study was to evaluate mosquito abundance, species diversity, larval and adult population dynamics in seven lagoons integrated in the wetland coastal system of the Algarve, Portugal, in the summer of 2007, as well as the screening of these for West Nile virus (WNV). WNV has been isolated from mosquitoes in this region, in the summer of 2004, next to the putative area of infection of two linked human WN cases. Adult mosquitoes were collected with CDC traps baited with CO(2), and potential breeding sites were surveyed for immature stages. Morphological identification of 1,432 adult mosquitoes and 85 larvae revealed the presence of 10 species: Anopheles atroparvus, Anopheles algeriensis, Coquillettidia richiardii, Culex modestus, Culex pipiens, Culex theileri, Culex univittatus, Culiseta longiareolata, Aedes caspius, and Aedes detritus. Adult mosquito peak densities were recorded in July, contrasting with null larval breeding in the same month in the surveyed biotopes. Most abundant species were C. pipiens (52%), C. theileri (29%), and A. caspius (11%). Lagoon Salgados and Quinta das Salinas, exhibited the highest similarity of culicid fauna, despite being most distant from each other, Female mosquitoes (1,249 specimens) screened by RT-PCR, did not reveal WNV products. However, previous detection of WNV activity in this area, susceptible to re-introductions, demands for continued vigilance. publishersversion published

The circulation of West Nile virus in Portugal was assessed by serological surveys conducted during 2004–2010 in horses and birds. The detection of WNV antibodies in both species in all the years covered by the study as well as the presence of anti-WNV IgM in symptomatic horses that had not traveled outside the country, support the notion that WNV circulates in Portugal.

West Nile virus (WNV), a Flavivirus distributed most widely, is presenting lately variable epidemiological and ecological patterns, including an increasing virulence that has already caused over 1000 human deaths in USA. Currently, diagnosis of WNV is achieved mainly by enzyme-linked immunoassays (ELISAs) based on the use of inactivated whole WNV (iWNV) as antigen, although results have to be confirmed by plaque reduction neutralization tests (PRNTs). Expression of WNV envelope recombinant E (rE) protein and its usefulness as ELISA antigen are described. Production of rE was achieved upon infection of Trichoplusia ni insect larvae with a recombinant baculovirus. Once optimized, the rE-based ELISA was validated with a battery of mouse and equine sera characterized previously. Concordance with the iWNV-based ELISA used routinely was good (95%), as it was with the reference PRNT (90%), with specificity of 94.4% and sensitivity of 88.1%. Production of rE protein in insect larvae allows for an easy, low cost and quite large-scale yield of partially purified antigen which is suitable for serological diagnosis of WNV, without the need for manipulation of large quantities of infective virus. © 2010 Elsevier B.V.