Neuroinvasive Cases Research Articles

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.

Surveillance and Early Warning of West Nile Virus Lineage 2 Using Backyard Chickens and Correlation to Human Neuroinvasive Cases.

In 2010, a West Nile virus (WNV) epidemic was reported in Central Macedonia, Northern Greece, with 197 neuroinvasive disease (WNND) cases in humans. The following 3 years, WNV spreads to new areas of Greece and human cases reoccurred during the transmission periods. After the initial outbreak, a WNV surveillance system using juvenile backyard chickens was established in Central Macedonia (after the 2011 outbreak) and Eastern Macedonia-Thrace (after the 2012 outbreak). Sera were screened for the presence of antibodies against WNV using cELISA and serum neutralization test, to monitor the spread of WNV and to assess the correlation between the WNV point seroprevalence in chickens and the incidence rates of human WNND cases in the aforementioned areas. WNV seroprevalence in chickens was 10.4% (95% CI: 7-15) in Central Macedonia (2011) and 18.1% (95% CI: 14-23) in Eastern Macedonia-Thrace (2012). Seroprevalence in chickens and incidence rates of human WNND cases in Eastern Macedonia-Thrace were strongly positively correlated (ρ = 0.98, P = 0.005) at the regional unit level, with the incidence of WNND in humans increasing with increasing WNV point seroprevalence in chickens. In Central Macedonia, the correlation was weaker (ρ = 0.68, P = 0.20), apparently due to small number of reported human WNND cases. Another study was also conducted using juvenile backyard chickens in Central Macedonia, aiming to detect early WNV enzootic circulation, before the onset of human cases during 2011 and 2013. The first seroconverted chickens were detected about 1.5 months before the laboratory diagnosis of any human WNND cases in Central Macedonia, for both years. WNV surveillance, using juvenile backyard chickens, was reliable for the identification of areas with WNV enzootic and silent transmission, and for early warning. Timely diffusion of information to public health authorities facilitated the successful implementation of preparedness plans to protect public health.

Chikungunya Virus: A Major Emerging Threat

Chikungunya Virus: A Major Emerging Threat

Risk assessment for West Nile Virus in Northern Greece (2010-2013)

Since the large WN fever epidemic in Central Macedonia in 2010 caused by WNV Lineage 2, with a total of 262 cases (197 neuroinvasive, incidence rate 1:140), WNV cases are reported every year from different areas throughout Greece (2011: 100 cases/75 neuroinvasive, 2012: 161/109, 2013: 86/51). WN fever has become one of the most important issues for the National Health Authorities in terms of vector-borne diseases. From 2011 and onwards, for the surveillance of WNV in Central Macedonia, two major networks were established by Ecodevelopment in collaboration with the Hellenic CDC, the region of Central Macedonia and four specialized laboratories: 1) A network of 60 CO2-traps for adult mosquitoes at fixed sites monitored biweekly for 4 months every year. Pools of 10-50 Culex spp. are forwarded weekly to the laboratories for the detection of WNV. 2) A network of sentinels (domestic pigeons and/or backyard chickens, 40 -50 hencoops or pigeon coops, 400-450 samples/year) for blood sampling in early summer and/or at the end of the hot season. In the plain of Thessaloniki, in early summer (June) the seroconversion in chickens reached 11,9% (28 positive/236 chickens) in 2011 versus 4,1% (8 positive/197 chickens) in 2013. The corresponding average weekly Minimum Infection Rate (M.I.R.) for the period mid June-end August was 1.73 in 2011 and 0.39 in 2013 respectively. These data seem to support the hypothesis that it is possible to relate the level of WNV circulation (infected mosquitoes and animal sentinels) with the upcoming human WNV cases: 16 human neuroinvasive cases were recorded in 2011 versus 5 cases in 2013. The follow up of the epidemiological risk through these two networks in combination with the weekly epidemiological reports of the Hellenic CDC and the results of larval habitat monitoring are permanently used to optimize vector control measures that are implemented in the region of Central Macedonia. Funding: Ecodevelopment, Hellenic Center for Disease Control & Prevention, Integrated Surveillance and Control Programme for West Nile Virus and Malaria in Greece

A Review of Vaccine Approaches for West Nile Virus

The West Nile virus (WNC) first appeared in North America in 1999. The North American lineages of WNV were characterized by the presence of neuroinvasive and neurovirulent strains causing disease and death in humans, birds and horses. The 2012 WNV season in the United States saw a massive spike in the number of neuroinvasive cases and deaths similar to what was seen in the 2002–2003 season, according to the West Nile virus disease cases and deaths reported to the CDC by year and clinical presentation, 1999–2012, by ArboNET (Arboviral Diseases Branch, Centers for Disease Control and Prevention). In addition, the establishment and recent spread of lineage II WNV virus strains into Western Europe and the presence of neurovirulent and neuroinvasive strains among them is a cause of major concern. This review discusses the advances in the development of vaccines and biologicals to combat human and veterinary West Nile disease.

Increased frequency of Tim-3+ T cells is associated with symptomatic WNV disease clinical outcome (P6085)

Abstract After a five-year period of steady annual cases, the 2012 West Nile virus (WNV) season was marked by a 4-fold resurgence with 5387 diagnosed infections, 2734 neuroinvasive cases and 243 deaths. Currently, no treatment or approved human vaccine is available for WNV infection. Understanding factors contributing to anti-WNV immunity and protection is therefore mandated. The present study investigates the surface expression patterns of negative T cell regulatory molecules over the course of WNV infection in a cohort of 32 WNV+ blood donors enrolled in longitudinal follow-up after initial WNV RNA+ blood donation (index) in those with asymptomatic (n=24) or symptomatic (n=8) disease outcome. We observed increased frequencies of Tim-3+CD4+ T cells during acute WNV infection (<90 days post-index) compared to uninfected controls (GEE P<0.0001). WNV+ donors had elevated frequencies of Tim-3+PD-1+ events in CD4+ and CD8+ T cells (P<0.05). Frequencies of Tim-3+ events in CD4+ and CD8+ T cells were higher in symptomatic than in asymptomatic subjects in the acute phase of infection (P=0.0009 and 0.033 on day 14 post-index, respectively), and symptomatic subjects had higher frequencies of Tim-3+CD4+ T cells throughout infection (GEE P<0.005). Our study highlights key mechanisms of immune vulnerability and reveals that T cell negative regulation is linked to symptomatic WNV disease and may serve as a novel target for improving protective responses or dampening inflammation and symptoms.

West Nile Virus Lineage 2 from Blood Donor, Greece

To the Editor: West Nile virus (WNV) is a mosquito-borne flavivirus that primarily causes an asymptomatic or mild disease in humans; however, in <1% of infected persons, it causes neurologic disease. The virus has received increased attention since 2002 when it was established that WNV is transmissible by blood transfusion and organ transplantation (1). A major WNV outbreak occurred in 2010 in Greece; most cases occurred in the northern part of the country (2). Of the 197 WNV neuroinvasive cases reported, 33 were fatal (3). Many nonneuroinvasive cases were observed (4). A lineage 2 WNV (Nea Santa-Greece-2010 strain) was detected in Culex pipiens mosquitoes collected at 2 locations where WNV cases had been reported (5). Although this strain shows high genetic identity to a Hungarian WNV strain isolated from birds in 2004, it has the amino acid substitution H249P in nonstructural protein 3 (NS3) (6). This mutation has been associated with increased virulence in WNV lineage 1 strains (7). Clinical WNV disease in humans had not been previously documented in Greece, and surveillance of blood donors in 2006 and 2007 did not show any WNV-positive result (8). On August 11, 2010, shortly after confirmation of the outbreak of WNV infections in humans in Greece, an action plan for the protection of blood safety was initiated. All donors were asked to report any fever-like illness up to 15 days after donation. Individual donation nucleic acid testing (NAT) of all blood donors living in the WNV-affected areas was implemented on August 22, 2010. The first WNV-positive by NAT result was obtained from a sample donated on August 22. Testing was performed by using the automated Procleix TIGRIS System (Chiron Corporation, Emeryville, CA, USA). The WNV-positive blood donor was a 40-year-old immunocompetent woman, a resident of a village in northern Greece. The village is located between 2 lakes, and the area is one of Europe’s major wetlands. The 2 locations where the WNV-positive mosquitoes were collected are near each other (Figure A1). The woman was working in an open-air fish market and reported numerous mosquito bites. At the time of blood donation, she was asymptomatic; 2 days later she had myalgia, arthralgia, and severe retro-orbital pain, lasting 2–3 days each. A second blood sample taken on August 26 was also NAT positive. Serologic testing for WNV IgM and IgG was performed by ELISA (WNV IgM Capture DxSelect and WNV IgG DxSelect; Focus Diagnostics Inc., Cypress, CA, USA). An index ≥1.5 for IgM and ≥1.1 for IgG was considered positive. No antibodies were detected in the initial and second serum samples; however, a third sample taken on September 20 was positive for WNV IgM and IgG (indices 4.7 and 3.8, respectively). Nested reverse transcription PCRs with the initial blood sample gave positive results (9,10). WNV lineage 2 sequences were obtained and were identical to those of the Nea Santa-Greece-2010 strain (6). One milliliter of each 1:10 and 1:100 dilution of whole blood in minimum essential medium containing antimicrobial drugs and 2% fetal bovine serum was placed on Vero E6 cell monolayers in 25-cm2 cell culture flasks. The procedure was performed in a biosafety level 3 laboratory, in which WNV lineage 2 had never been handled. After the sample was incubated 1 hour at 37°C in a 5% CO2 incubator, 9 mL of fresh medium containing 4% fetal bovine serum was added. Cytopathic effects were observed in the flask inoculated with the 1:100 dilution on day 3 after infection. An aliquot of supernatant was used to infect fresh cell monolayers. WNV growth in the cell culture was demonstrated by reverse transcription PCR and immunofluorescence assay. Sequences of the cell culture isolate were identical to those of the directly detected virus. To check whether the isolate possessed the H249P substitution, a set of nested primers spanning the region 5140–5660 from the WNV genome was designed: NS3a-5′-GCTGGCTTCGAACCTGA-3′ and NS3b-5′-CAATCATCGTTCTTGC-3′ for the first round PCR and NS3c-5′-GCTGCTGAGATGTCTGA-3′ and NS3d-5′-TCATATCCAGTGTTCCA-3′ for the nested PCR. The H249P substitution was present. Sequences were submitted to GenBank (accession nos. {type:entrez-nucleotide,attrs:{text:JF917091,term_id:349806861}}JF917091, {type:entrez-nucleotide,attrs:{text:JF917092,term_id:349806863}}JF917092). Virus isolation from WNV patients is usually unsuccessful because viremia levels are low and last only a short time. WNV strains are usually isolated from immunocompromised patients, blood donors, IgM-negative immunocompetent patients who seroconverted, or autopsy brain samples. For the donor reported here, WNV was isolated 2 days before illness onset, when no antibodies were present. The WNV-positive blood donor was detected 1 day after the introduction of blood screening. The early diagnosis of the initial human WNV cases in Greece, which resulted in prompt implementation of NAT testing, had a substantial positive impact on the safety of the blood supply in the affected areas. The risk for virus transmission was reduced for blood recipients, in particular those who receive multiple transfusions and immunocompromised patients in need of transfusion.

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.

Development time of IgG antibodies to West Nile virus

Following an outbreak of West Nile virus (WNV) infections in Greece during summer/autumn 2010, a study was conducted to investigate the patterns of WNV IgG reactivity in 255 patients with respect to the day of illness and the type of clinical syndrome. IgG antibodies were detectable after a mean of 8.1 ± 4.9 and 12.6 ± 11.3 days after onset of illness in neuroinvasive and non-neuroinvasive cases, respectively (p < 0.001), suggesting that a delay in the development of WNV IgG antibodies is seen in non-neuroinvasive cases.

Simulation of the seasonal cycles of bird, equine and human West Nile virus cases

The West Nile virus (WNV) is an arthropod-borne virus (arbovirus) circulating in a natural transmission cycle between mosquitoes (enzootic vectors) and birds (amplifying hosts). Additionally, mainly horses and humans (dead-end hosts) may be infected by blood-feeding mosquitoes (bridge vectors). We developed an epidemic model for the simulation of the WNV dynamics of birds, horses and humans in the U.S., which we apply to the Minneapolis metropolitan area (Minnesota). The SEIR-type model comprises a total of 19 compartments, that are 4 compartments for mosquitoes and 5 compartments or health states for each of the 3 host species. It is the first WNV model that simulates the seasonal cycle by explicitly considering the environmental temperature. The latter determines model parameters responsible for the population dynamics of the mosquitoes and the extrinsic incubation period. Once initialized, our WNV model runs for the entire period 2002–2009, exclusively forced by environmental temperature. Simulated incidences are mainly determined by host and vector population dynamics, virus transmission and herd immunity, respectively. We adjusted our WNV model to fit monthly totals of reported bird, equine and human cases in the Minneapolis metropolitan area. From this process we estimated that the proportion of actually WNV-induced dead birds reported by the Centers for Disease Control and Prevention is about 0.8%, whereas 7.3% of equine and 10.7% of human cases were reported. This is consistent with referenced expert opinions whereby about 10% of equine and human cases are symptomatic (the other 90% of asymptomatic cases are usually not reported). Despite the restricted completeness of surveillance data and field observations, all major peaks in the observed time series were caught by the simulations. Correlation coefficients between observed and simulated time series were R = 0.75 for dead birds, R = 0.96 for symptomatic equine cases and R = 0.86 for human neuroinvasive cases, respectively.

West Nile Virus Encephalitis in a Patient with Renal Transplant

Introduction Most cases of West Nile Virus infection are asymptomatic. 60% to 70% of neuro-invasive cases of West Nile virus infection result in meningitis or encephalitis; however, West Nile encephalitis occurs in less than 1% of patients infected with the West Nile virus.1

Human West Nile Virus Infection, Catalonia, Spain

To the Editor: West Nile virus (WNV) is a mosquitoborne flavivirus that is widespread in Africa, the Middle East, Asia, and southern Europe, where it causes outbreaks and sporadic cases of the disease. It has become an emergent disease in North America, where it was detected for the first time in 1999 and became epidemic shortly thereafter (1). Although WNV was initially considered to have a minor health effect in the Mediterranean basin, human and equine outbreaks reported in the last decade in different countries (2–5) have made WNV infections a public health concern. The epidemiology of WNV in Europe differs from that in America and has only been associated with nonrecurrent, sporadic outbreaks. The reasons for this difference are controversial; it may be due to environmental factors, reservoirs, or even mosquito vectors. In Spain, neither equine nor human WNV cases have been reported. However, some human serosurveys that used hemagglutination inhibition suggested that WNV or closely related flaviviruses circulated during the 1970s in the Ebro delta and areas in Spain (6,7). The Ebro delta, a wetland in Catalonia, in the northeast of Spain, is a stopping-off point for birds migrating between regions of Africa and Europe where different WNV vectors and reservoirs have been identified. The delta could be considered a high-risk area for WNV and other arthropodborne virus infections. To evaluate WNV seroprevalence in the human population of the Ebro delta, a survey was conducted in 2001. After obtaining informed consent, 992 serum samples were obtained from inhabitants of the area. The population studied was representative of the whole area and was stratified by sex and age. Anti-WNV immunoglobulin G (IgG) antibodies were determined by using an in-house indirect enzyme-linked immunosorbent assay (ELISA), as previously described (8). Results were classified as the sample absorbance/positive control absorbance ratio. Samples showing ratio values >0.2 were tested for WNV IgG and IgM by using an indirect and a μ-chain capture ELISA, respectively (Focus Technologies, Cypress, CA, USA), and an in-house microneutralization test. For the microneutralization test, samples were tested in duplicate and assayed twice. Twofold dilutions (25 μL) of the samples (1:16–1:256 dilutions) were assayed by using 100 TCID50 (50% tissue culture infectious dose) of West Nile Eg-101 reference strain in 96-well tissue culture plates with Vero cells and after 7 days of incubation at 37°C and 5% CO2. Thirty-eight samples showed IgG ratios >0.2 by the in-house ELISA. Of these, 12 showed WNV IgG, and 1 was positive for WNV IgM and IgG, according to the Focus assays. Two samples showed positive neutralizing activity, with titers of 32 and 256. The highest titer was shown by the sample that yielded positive levels of both IgM and IgG in the ELISA, which suggests recent WNV infection. Anti-WNV IgG was more often detected in participants in the 20- to 29-year age group (odds ratio [OR] 4.23, 95% confidence interval [CI] 1.04–16.02, p = 0.03) and in persons who reported frequent mosquito bites (OR 8.62, 95% CI 0.44–169, p = 0.08). IgG-positive persons were equally divided by sex. No significant differences were found between antibody-positive or antibody-negative persons with respect to their profession, place of occupation, current residence, time in current residence, outdoor activities, use of insecticides and repellents, or symptoms related to WNV infection. No symptoms related to WNV infection were reported by the IgM/IgG-positive participant, who was 31 years of age, was born in the area, worked outdoors, and was frequently bitten by mosquitoes. He also reported travel to Cuba 1 year earlier, but he had not been vaccinated against flavivirus, and serologic test results for dengue were negative. The other IgG- and neutralizing antibody–positive participant was 45 years of age and was born and works in the area. He had never traveled abroad or been vaccinated against flavivirus. He reported a 4-day fever of unknown origin during the summer 1 or 2 years before the study. He often fishes in the areas and is frequently bitten by mosquitoes. In conclusion, the study found evidence of recent WNV infections in humans living in the Ebro delta, where previous flavivirus circulation has been suggested by Lozano and Filipe (6). IgG-positive results not confirmed by neutralization could be due to cross-reactive antibodies induced by other flavivirus infections or vaccinations (9,10). The probable WNV infection described was asymptomatic, as occurs in ≈80% of cases. Other WNV infections in the area may have remained undetected, including neuroinvasive cases. Intensified research and surveillance in this area will help determine and refine thresholds for public health interventions.