Comparison of Stable and Transient Wolbachia Infection Models in Aedes aegypti to Block Dengue and West Nile Viruses.

Pathogen replication and transmission in Wolbachia infected insects are currently studied using three Wolbachia infection systems: naturally infected Wolbachia hosts, hosts transinfected with Wolbachia (stably maintained and inherited infections) and hosts transiently infected with Wolbachia. All three systems have been used to test the effect of Wolbachia on mosquito transmitted pathogens such as dengue virus (DENV), West Nile virus (WNV) and Plasmodium. From these studies it is becoming increasingly clear that the interaction between a particular pathogen and Wolbachia is heavily influenced by the host-Wolbachia interaction and the model of infection. In particular, there is some evidence that under very specific conditions, Wolbachia can enhance pathogen infection in some hosts. In this study, we compared the effect of Wolbachia in two infection models (stable transinfected and transiently infected) on the replication, infection- and transmission rates of two flaviviruses, DENV and WNV (Kunjin strain). Our results indicate that Wolbachia had similar blocking effects in both stable and transient models of infection, however, the magnitude of the blocking effect was significantly lower in mosquitoes transiently infected with Wolbachia. More importantly, no evidence was found for any enhancement of either DENV or WNV (Kunjin strain) infection in Ae. aegypti infected with Wolbachia, supporting a role for Wolbachia as an effective and safe means for restricting transmission of these viruses.

First detection of West Nile virus in domestic pigeon in Korea.

West Nile virus (WNV) is a mosquito-borne zoonotic pathogen that has spread throughout Europe and the United States. Recently, WNV spread to East and Southeast Asia, and great efforts have been made in South Korea to prevent the spread of WNV from neighboring countries. In this study, we diagnosed the first case of WNV in pigeons (Columba livia domestica) residing in cities using a competitive enzyme-linked immunosorbent assay and confirmed it with nested reverse transcription polymerase chain reaction analysis and sequencing. This is the first report to provide convincing evidence that WNV is present within South Korea.

Epidemiology of West Nile disease in Europe and in the Mediterranean Basin from 2009 to 2013.

West Nile virus (WNV) transmission has been confirmed in the last four years in Europe and in the Mediterranean Basin. An increasing concern towards West Nile disease (WND) has been observed due to the high number of human and animal cases reported in these areas confirming the importance of this zoonosis. A new epidemiological scenario is currently emerging: although new introductions of the virus from abroad are always possible, confirming the epidemiological role played by migratory birds, the infection endemisation in some European territories today is a reality supported by the constant reoccurrence of the same strains across years in the same geographical areas. Despite the WND reoccurrence in the Old World, the overwintering mechanisms are not well known, and the role of local resident birds or mosquitoes in this context is poorly understood. A recent new epidemiological scenario is the spread of lineage 2 strain across European and Mediterranean countries in regions where lineage 1 strain is still circulating creating favourable conditions for genetic reassortments and emergence of new strains. This paper summarizes the main epidemiological findings on WNV occurrence in Europe and in the Mediterranean Basin from 2009 to 2013, considering potential future spread patterns.

Wolbachia enhances West Nile virus (WNV) infection in the mosquito Culex tarsalis.

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.

Linking mosquito infestation to resident socioeconomic status, knowledge, and source reduction practices in suburban Washington, DC.

Eliminating water-holding containers where mosquitoes oviposit and develop (source reduction) can help manage urban disease-vector mosquitoes. Source reduction requires residents to be knowledgeable of effective practices and motivated to implement them. We tested relationships between demographics, resident knowledge, attitudes, and practices (KAP), and mosquito infestation by administering larval mosquito surveys and KAP questionnaires in Washington, DC. Respondents who reported practicing source reduction had lower numbers of pupae-positive containers and Culex pipiens-positive containers, but not Aedes albopictus-positive containers or water-holding containers, in their yards. When controlling for numbers of water-holding containers in statistical models, residents who reported source reduction had lower numbers of A. albopictus-positive containers in addition to numbers of pupae-positive containers and C. pipiens-positive containers. These results suggest that while active container reduction may be effective at reducing C. pipiens and overall pupal production, it may be offset by other resident activities that add containers to yards, and that source reduction that involves mosquito habitat management without outright container removal can also be effective at reducing A. albopictus. Source reduction was related to respondent knowledge of mosquitoes and, in particular, specific knowledge of mosquito development, which both varied with demographics alongside respondent motivation to control mosquitoes. Respondents from high socioeconomic status households reported greater knowledge but lower motivation than respondents from middle and low socioeconomic-status households. We conclude that mosquito-related education will help promote community-based container management as part of integrated mosquito management programs, particularly in middle and low socioeconomic status neighborhoods with lower knowledge and high motivation.

Emergence of West Nile virus infections in humans in Turkey, 2010 to 2011.

In 2010, 47 human cases of West Nile virus (WNV)infection, including 12 laboratory-confirmed and 35 probable cases, were identified in Turkey. These were the first cases detected during routine surveillance.The patients were from 15 provinces, mainly located in the western part of the country. Incidence was 0.19/100,000 with a maximum of 1.39 in Sakarya province.Forty of the total 47 cases showed neuroinvasive manifestation. Median age was 58 years with a range of four to 86. Ten of the patients died. Enhanced surveillance in humans and animals and mosquito control measures were implemented. The WNV infections were included in the national notifiable diseases list as of April 2011. In 2011, three probable and two confirmed cases of WNV infection were diagnosed in provinces where infections had been detected in the previous year, supporting a lower activity than 2010. However,detection of WNV infections in humans in 2010 and 2011 consecutively, may indicate that WNV has become endemic in the western part of Turkey. Field epidemiological studies were undertaken to understand more about the nature of infection in Turkey.

The native Wolbachia endosymbionts of Drosophila melanogaster and Culex quinquefasciatus increase host resistance to West Nile virus infection.

BACKGROUND: The bacterial endosymbiont Wolbachia pipientis has been shown to increase host resistance to viral infection in native Drosophila hosts and in the normally Wolbachia-free heterologous host Aedes aegypti when infected by Wolbachia from Drosophila melanogaster or Aedes albopictus. Wolbachia infection has not yet been demonstrated to increase viral resistance in a native Wolbachia-mosquito host system. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated Wolbachia-induced resistance to West Nile virus (WNV; Flaviviridae) by measuring infection susceptibility in Wolbachia-infected and Wolbachia-free D. melanogaster and Culex quinquefasciatus, a natural mosquito vector of WNV. Wolbachia infection of D. melanogaster induces strong resistance to WNV infection. Wolbachia-infected flies had a 500-fold higher ID50 for WNV and produced 100,000-fold lower virus titers compared to flies lacking Wolbachia. The resistance phenotype was transmitted as a maternal, cytoplasmic factor and was fully reverted in flies cured of Wolbachia. Wolbachia infection had much less effect on the susceptibility of D. melanogaster to Chikungunya (Togaviridae) and La Crosse (Bunyaviridae) viruses. Wolbachia also induces resistance to WNV infection in Cx. quinquefasciatus. While Wolbachia had no effect on the overall rate of peroral infection by WNV, Wolbachia-infected mosquitoes produced lower virus titers and had 2 to 3-fold lower rates of virus transmission compared to mosquitoes lacking Wolbachia. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that Wolbachia can increase resistance to arbovirus infection resulting in decreased virus transmission in a native Wolbachia-mosquito system. The results suggest that Wolbachia reduces vector competence in Cx. quinquefasciatus, and potentially in other Wolbachia-infected mosquito vectors.

Seroprevalencia de infecciones por el virus del Nilo Occidental en la población general del sur de España / West Nile virus past infections in the general population of Southern Spain

Objective. To analyze the prevalence of past and recent infections by West Nile virus (WNV) and the risk factors associated with WNV exposure in a representative population from southern Spain. Methods. Sample size was established for an estimated prevalence of past WNV infections of 5 6 2.5% in 504 subjects. A pre-stratification was performed according to age distribution and place of residence. After random telephone solicitation and acquisition of informed consent, a serum sample was collected and an epidemiologic survey performed on all participating subjects. Samples were tested with ELISA-IgG and MAC-ELISA to detect specific IgG and IgM antibodies; results were confirmed by the plaque reduction neutralization test (PRNT). Multivariate analysis using a forward stepwise logistic regression model was performed to assess potential risk factors associated with WNV exposure. Results. Prevalence of past WNV infections confirmed by PRNT in the 504 participants was 0.6%, affecting mainly older persons (mean age 65 6 23 vs. 34 6 22 years; P 5 0.018), those living in rural areas (5.4% vs. 0% in urban areas; P 5 0.01), and individuals with risk professions (prevalence 2.8% vs. 0%; P 5 0.048). None of the five recent infections detected by MAC-ELISA was confirmed by PRNT. Conclusions. These results strongly suggest past circulation and exposure of the human population to WNV in southern Spain (AU)

Objetivo. Analizar la prevalencia de infecciones pasadas y recientes por el virus del Nilo Occidental (VNO), así como los factores de riesgo asociados con la exposición al mismo, en una muestra representativa de la población del sur de España. Métodos. El tamaño de la muestra se estableció para una seroprevalencia de infección pasada del 5 6 2,5% en 504 sujetos, preestratificándose ésta por edad y lugar de residencia. Los voluntarios se incluyeron tras solicitud telefónica y consentimiento, y a todos ellos se les realizó una extracción de suero y una encuesta epidemiológica. Las muestras se analizaron para detectar IgG e IgM específicas, mediante enzimoinmunoensayo (ELISA-IgG) y enzimoinmunoensayo por captura (MAC-ELISA), confirmando ulteriormente los resultados positivos mediantereducción-neutralización en placa (PRNT. Finalmente se realizó un análisis de regresión logística multivariante paso a paso hacia delante para determinar los posibles factores asociados con la exposición al VNO. Resultados. La prevalencia de infecciones por VNO confirmadas por PRNT en los 504 sujetos incluidos fue del 0,6%, y se vieron principalmente afectadas las personas de mayor edad (edad media 65 6 23 frente a 34 6 22 años; p 5 0,018), las que vivían en áreas rurales (prevalencia del 5,4% frente al 0% en áreas urbanas; p 5 0,01), y las personas con profesiones de riesgo (prevalencia del 2,8% frente al 0%; p 5 0,048). Por el contrario, ninguna de las infecciones recientes detectadas por MAC-ELISA fue confirmada ulteriormente por PRNT. Conclusiones. Estos resultados apoyan firmemente la circulación en el pasado del VNO, así como la exposición humana del mismo, en áreas del sur de España (AU)

Medical devices; immunology and microbiology devices; classification of the West Nile Virus IgM capture Elisa assay. Final rule.

The Food and Drug Administration (FDA) is classifying the West Nile Virus IgM Capture Elisa assay into class II (special controls). The agency is taking this action in response to a petition submitted under the Federal Food, Drug, and Cosmetic Act (the act) as amended by the Medical Device Amendments of 1976 (the amendments), the Safe Medical Devices Act of 1990, and the Food and Drug Administration Modernization Act of 1997 (FDAMA). The agency is classifying this device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of a guidance document that will serve as the special control for the device.

[Search for inhibitors of West Nile virus among antibiotics]. / Poisk ingibitorov virusa Zapadnogo Nila sredi antibiotikov.

The activity of 24 antibiotics was studied in treatment of albino mice with experimental encephalitis caused by West Nile virus. The antiviral activity of gentamicin and kanamycin was stated. The survival rate of the animals 19. contaminated with 10-100 LD50 of the West Nile virus and treated parenterally with gentamicin in a dose of 80 to 400 micrograms/mouse was higher than that in the controls by 29.5 to 100 per cent and depended on the drug regimen. The efficacy of kanamycin was lower. The chemotherapeutic indices of gentamicin and kanamycin amounted to 100 and 10, respectively. Since there are no schemes for chemotherapy of the infection caused by the West Nile virus and the respective vaccines are not available the use of the antibiotics and gentamicin in particular appears to be promising in the disease prevention and treatment.

Neuronal cell cultures as a model for assessing neurotoxicity induced by encephalitic viruses.

Primary dispersed and organotypic cultures were prepared from selected brain areas and spinal cords of rat (Sprague-Dawley) and mouse (SJL/OLA(F) Ness-Ziona) fetuses and neonates. Following fiber regeneration, synapse formation and myelination, cultures were infected with one of the following viruses: Rabies CVS-21 strain, Sindbis Alphavirus, West-Nile Flavivirus and Theiler Murine Encephalomyelitis virus. Light and electron microscopical studies showed clear differences in the target cells for virus infection; time of viral replication and in the intensity and specificity of the cytopathic effects induced by these viruses. Thus, Sindbis and Theiler viruses induced severe cytotoxicity and demyelination due to rapid viral replication in both neurons and all glial cell types. Rabies and West-Nile viruses, on the other hand, replicated mainly in neurons and at a much slower rate, causing only mild damage to the cells and the myelin sheath. A very specific alignment of West-Nile virions was observed along the interperiod lines of the myelin sheath in several myelinated axons. This peculiar arrangement of the virions, entrapped between the myelin lamellae may lead to a novel concept in the understanding of viral infection.

Protection by dehydroepiandrosterone in mice infected with viral encephalitis.

Dehydroepiandrosterone (DHEA) has a significant protective effect in mice infected with West Nile virus (WNV), Sindbis virus neurovirulent (SVNI) and Semliki Forest virus (SFV). Mice injected subcutaneously (SC) with a single injection of DHEA (1 g/kg) on the same day or one day pre or post infection with WNV resulted in 40-50% mortality as compared to 100% in control injected mice (p less than 0.05). The drug was effective following a single SC injection or serial intraperitoneal (IP) injections (5-20 mg/kg) on days 0, 2, 4, and 6 following virus inoculation. Moreover, DHEA injection not only reduced viremia and death rate, but also significantly delayed the onset of the disease and mortality. The titers of antivirus antibodies in surviving mice were very high. However, DHEA had no effect on WNV growth in BHK or Vero cell cultures. In this study it was shown that DHEA protects mice against WNV, SVNI and SFV lethal infection. Though the mechanism of the protective effect of DHEA is still unknown, it seems that DHEA can modify the host resistance mechanisms rather than the virus itself.

Comparative analysis of West Nile virus strains isolated from human and animal hosts using monoclonal antibodies and cDNA restriction digest profiles.

Three West Nile (WN) virus strains isolated in Bangui, Central African Republic (CAR), from patients with hepatitis were analysed comparatively with the prototype WN virus strain and 7 WN strains previously isolated from birds (2 strains), mosquitoes (3 strains) and ticks (2 strains) in CAR. The comparison was based on two techniques: an epitopic analysis by indirect immunofluorescence assay using a panel of 9 monoclonal antibodies to WN virus, and an analysis of HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA. Similar results were obtained with both techniques: the 3 human strains were found to be identical to each other and identical or very close to mosquito and tick strains, whereas prototype WN virus and bird strains were significantly different from the human strains. As "classical" infections due to WN virus without hepatic involvement were also reported during the period of isolation of the arthropod strains, we concluded that the same virus subtype may have been the cause of different infection patterns. A new definition of the disease spectrum of WN virus, including the possibility of liver involvement, should be established. Clearly, the Egyptian prototype WN virus represents a different topotype. Bird strains also appear to be different from human and arthropod strains, raising the question of their transmissibility and pathogenicity for man, and of the role of birds in the natural cycle of WN virus.

The influence of cold or isolation stress on resistance of mice to West Nile virus encephalitis.

The effect of cold or isolation stress on mortality rate and brain virus level were investigated in mice infected with West Nile virus (WNV). Exposure of mice for 5 min/day to cold water (1 +/- 0.5 degrees C) for 8-10 days resulted in 92% mortality as compared to 47% in control mice (p less than 0.001). Mice housed in individual cages (isolation stress) were also more susceptible to WN viral infection, as shown by increased mortality rate reaching 85% as compared to 50% in mice housed 6 per cage (p less than 0.01). Cold or isolation stress increased blood brain and spleen virus levels as early as 2 days after inoculation. After 8 days of isolation or cold stress, mice inoculated with WNV had 8.9 and 9.0 log10 plaque forming units in the brain, respectively, as compared to 6.9 in the control (p less than 0.01-0.001). Furthermore, lymphoid organs such as spleen and thymus showed severe mass loss. These data suggest that physical or non-physical stress situations enhance WNV encephalitis by accelerating virus proliferation and increase mortality in mice.

The influence of cold or isolation stress on neuroinvasiveness and virulence of an attenuated variant of West Nile virus.

The effect of cold or isolation stress on neuroinvasiveness and virulence was investigated in mice inoculated with an attenuated WNV (WN-25) strain. The WN-25 variant differed from the parent strain by its inability to kill mice after I.P. injection though it was able to immunize even after injection with low doses of virus. Exposure of inoculated mice for 5 minutes a day to cold water (1 +/- 0.5 degrees C) for 8 days resulted in 60% mortality, while in nonstressed infected mice no death was observed. Cold or isolation stress increased the virus level in the brain to 8.9 and 7.4 log 10 PFU as compared to no virus in the infected control. Moreover, it was found that virus level in the spleen of stressed mice reached 3.4 and 3.7 log 10 PFU respectively, while in non-stressed mice no virus was detected. The virus which was isolated from the brain of moribund stressed mice was extremely virulent: I.P. inoculation of as little as 10 PFU caused death to normal non-stressed mice. We suggest that cold or isolation stress conditions in mice inoculated with an attenuated strain induce a selection process. The virus which was isolated from the brain of stressed mice changes its virulence and kills like wild type WNV.

Absence of viraemia in cattle after experimental infection with Japanese encephalitis virus.

Cow calves were infected with Japanese encephalitis virus (JEV) by parenteral inoculation. One batch was reinfected with JEV, followed by West Nile virus (WNV), while another batch was reinfected directly with WNV. No viraemia due to either JEV or WNV was demonstrated in any of the calves. Culex tritaeniorhynchus mosquitoes fed on 4 of the calves infected with JEV during the first 10 d had no detectable virus, nor did they transmit the virus by bite to susceptible baby chickens. In another experiment, calves did not develop viraemia after infected C. tritaeniorhynchus mosquitoes were allowed to feed on them. Neutralizing and/or haemagglutination-inhibiting antibodies against JEV were demonstrated in 6 of the 11 calves, which explains the high proportion of JE seropositives among cattle in India. All the 5 calves that were infected with WNV subsequent to JEV developed neutralizing and haemagglutination-inhibiting antibodies against WNV also. The study indicates that cattle do not play a role in the maintenance of JEV in nature.

Arboviruses and lemurs in Madagascar: experimental infection of Lemur fulvus with yellow fever and West Nile viruses.

In previous serological surveys of lemurs in Madagascar, antibodies against flaviviruses were frequently detected. To examine the epidemiological role of Lemur fulvus, experimental infections with yellow fever (YF) virus and West Nile (WN) virus were performed. YF and WN infections were clinically unapparent. A 3 to 4-day-long viremia, with moderate levels was observed with YF virus. WN virus, especially the strain isolated in Madagascar, provoked a 4 to 6-day-long viremia sufficient to infect Aedes aegypti. In all experiments, the antibody response was studied during the following weeks by 3 methods. The results led to the conclusion that Malagasy lemurs could act as amplifying hosts for WN virus present in Madagascar, and as hosts for YF virus if it were introduced on the island. The epidemiological role of these primates is discussed according to their ecology and their contact with potential mosquito vectors in forest areas of Madagascar.