Circulation Of Flaviviruses Research Articles

Flaviviruses in Europe: complex circulation patterns and their consequences for the diagnosis and control of West Nile disease.

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.

Recent Circulation of West Nile Virus and Potentially Other Closely Related Flaviviruses in Southern France

In recent years, the number of West Nile virus (WNV) cases reported in horses and humans has increased dramatically throughout the Mediterranean basin. Furthermore, the emergence of Usutu virus (USUV) in Austria in 2001, and its subsequent expansion to Hungary, Spain, Italy, Switzerland, the United Kingdom, and Germany, has given added cause for concern regarding the impact of the spread of flaviviruses on human and animal health in western Europe. Despite frequent detection of WNV and USUV cases in neighboring countries, no case of WNV has been detected in France since 2006 and USUV has never been reported. However, recent investigations focused on detecting the circulation of flaviviruses in France are lacking. We investigated the circulation of WNV and USUV viruses in wild birds in southern France on the basis of a serological survey conducted on a sentinel species, the magpie (Pica pica), in the Camargue area from November, 2009, to December, 2010. We detected WNV-neutralizing antibodies at a high titer (160) in a second-year bird showing recent exposure to WNV, although no WNV case has been detected in humans or in horses since 2004 in the Camargue. In addition, we observed low titers (10 or 20) of USUV-specific antibodies in six magpies, two of which were also seropositive for WNV. Such low titers do not give grounds for concluding that these birds had been exposed to USUV; cross-reactions at low titers may occur between antigenically closely related flaviviruses. But these results urge for further investigations into the circulation of flaviviruses in southern France. They also emphasize the necessity of undertaking epidemiological studies on a long-term basis, rather than over short periods following public health crises, to gain insight into viral dynamics within natural reservoirs.

Evidence of mosquito-transmitted flavivirus circulation in Piedmont, north-western Italy

BackgroundFlavivirus is a highly heterogeneous viral genus that includes important human pathogens and several viral strains with unknown zoonotic potential. Mosquito-borne flaviviruses have been isolated and characterized in Northern Italy: West Nile virus and Usutu virus were detected in mosquitoes and in different host species and recent studies provided evidence about the circulation of “insect Flavivirus” strains.MethodsIn order to clarify the diffusion and the distribution of the mosquito-transmitted flaviviruses in Italy, we analyzed Culex and Ochlerotatus mosquitoes collected in 2009 and 2010 in an area divided evenly between hills and plains and where the landscape is dominated by mixed agricultural patches, rice fields, deciduous tree forests, and urban environments. Each mosquito pool was tested for the presence of Flavivirus strains and we characterized positive samples by genetic sequencing.ResultsPositive mosquito pools revealed low infection prevalence, but suggested a continuous circulation of both Usutu virus and insect Flavivirus. Interestingly, phylogenetic analyses based on NS5 gene partial sequences showed a closer relationship among new Usutu virus strains from Piedmont and the reference sequences from the Eastern Europe, with respect to Italian samples characterized so far. Moreover, NS5 gene phylogeny suggested that mosquito flaviviruses found in Italy could belong to different lineages.ConclusionsOur results contribute to a wider point of view on the heterogeneity of viruses infecting mosquitoes suggesting a taxonomical revision of the Mosquito-borne Flavivirus group.

Do Wild Ungulates Allow Improved Monitoring of Flavivirus Circulation in Spain?

As a response to the need for improved and cost-efficient West Nile virus (WNV) and other flavivirus surveillance tools, we tested 887 juvenile free-living red deer, 742 free-living juvenile wild boar, and 327 farmed deer, to detect temporal variability in exposure to these viruses. Thirty of 742 wild boar samples (4%; 95% CI 2.8,5.7) yielded a positive ELISA result. Antibody-positive individuals had been sampled between 2003 and 2011 in localities from central and southern Spain. No wild boar from the northern half of Spain (n=120) tested positive. Regarding juvenile wild red deer, only two out of 887 samples yielded a positive ELISA result (0.2%; 95% CI 0.1,0.8). These two samples came from the same site and sampling year. The likelihood of detecting contact with WNV or cross-reacting flaviviruses was 18 times higher among juvenile wild boar than among juvenile red deer. ELISA positivity among farmed deer increased 10-fold after local flavivirus outbreaks recorded in the summer and autumn of 2010. This survey demonstrated the potential usefulness of juvenile wild ungulates, particularly wild boar, as suitable flavivirus sentinels in southwestern Europe, and that systematic serum banking of samples from hunter-harvested wildlife or from individual farmed ungulates provides valuable material for retrospective epidemiological surveys and future disease monitoring.

Serosurvey of West Nile Virus and Other Flaviviruses of the Japanese Encephalitis Antigenic Complex in Birds from Andalusia, Southern Spain

Flaviviruses of the Japanese encephalitis virus (JEV) antigenic complex, including West Nile virus (WNV), are recognized as emerging and reemerging pathogens. Circulation of flaviviruses has been recently detected in different mosquito and vertebrate species in several European countries. A serosurvey study was carried out to evaluate the circulation of WNV and other flaviviruses of the JEV antigenic complex in different wild bird species in Spain between 2006 and 2009. Seropositiviy against JEV using a competitive enzyme-linked immunosorbent assay was found in common coot, Montagu's Harrier, black kite, black vulture, Bonelli's eagle, Spanish imperial eagle, Egyptian vulture, and Eurasian spoonbill. Seropositivity to JEV antigenic complex viruses was significantly higher in samples collected during autumn compared with animals sampled during summer. Significantly higher seroprevalence was also observed in 2007 compared with 2009, whereas there were no significant differences in seropositivity among taxonomic levels, migratory versus resident behavior, body size (large vs. medium), or habitats (free-ranging vs. captivity). Neutralizing antibodies against WNV were detected in common coot and Spanish imperial eagle using a virus-neutralization test. Oral shedding of WNV was not detected in any of the Spanish imperial eagles, Egyptian vultures, Eurasian Spoonbills, Lammergeiers, and the Black vultures analyzed by means of real-time reverse transcription-polymerase chain reaction. The results indicate that WNV and others flaviviruses of the JEV antigenic group circulated in migratory and resident wild bird species in Spain between 2007 and 2008. Further studies are necessary to determine the precise role that each of these wild bird species, some of them cataloged as "near threatened," "vulnerable," or "endangered," play in the epidemiology of those viruses.

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.