Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.     

Odorant-Binding Protein from Culex tarsalis,the Most Competent Vector of West Nile Virus in California

We have identified and cloned an odorantbinding protein from the female mosquito, Culex tarsalis (CtarOBP). As expected for an olfactory protein, CtarOBP was detected by gel electrophoresis analysis in antennae but not in control tissues (legs). The isolated protein was identified by in-gel digestion and subsequent analysis of internal fragments by tandem mass spectrometry (MS-MS). Based on the amino acid sequences of two peptides generated by enzymatic digestion, degenerate primers were designed for cDNA cloning. The complete cDNA (cloned by RACE) encoded a protein with a signal peptide (24 residues) and a mature protein of 125 amino acid residues. The calculated molecular mass and isoelectric point of the mature protein were 14,515 Da and pl 5.5, respectively. CtarOBP showed the hallmark of odorant-binding proteins, 6 cysteine residues, and high sequence homology (61–96%) to previously characterized mosquito OBPs.     

West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance

Background

West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic.

Methodology/Principal Findings

We assessed WNV vector competence of Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase.

Conclusions/Significance

Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe.     

Small Interfering RNA-Mediated Control of Virus Replication in the CNS Is Therapeutic and Enables Natural Immunity to West Nile Virus

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West Nile Virus Seroprevalence in the Greek Population in 2013: A Nationwide Cross-Sectional Survey

Cases of West Nile Virus (WNV) disease were recorded for three consecutive years in Greece following the year 2010 outbreak. A cross-sectional serologic survey was conducted to estimate the WNV seroprevalence and assess the ratio of infection to neuroinvasive disease. A stratified left-over sampling methodology was used including age and residence strata. A total of 3,962 serum samples was collected and tested for WNV Immunoglobulin G (IgG) antibodies by Enzyme–Linked Immunosorbent Assay (ELISA). All positive samples were further tested by Plaque Reduction Neutralization Test (PRNT) and WNV Immunoglobulin M (IgM) antibodies. WNV IgG antibodies were detected in 82 samples and 61 were also positive in PRNT representing a weighted seroprevalence of 2.1% (95% C.I.: 1.7–2.6) and 1.5% (95% C.I.: 1.2–2.0), respectively. Multivariable analysis showed that seroprevalence was associated with age and residence. The overall ratio of neuroinvasive disease to infected persons was estimated at 1:376 (95% C.I.: 1:421–1:338), while the elderly people had the highest ratio. This nationwide study provided valuable data regarding the epidemiology of WNV in Greece based on the fact that elderly people have higher risk of being both infected and having severe disease.     

Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region

West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8) and 10(8.5) plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.     

Control of arbovirus infections by a coordinated response: West Nile Virus in England and Wales

Although there is no recognized transmission of human arboviral infections in the UK, concerns about the possible spread of West Nile virus (WNV) have precipitated coordinated activities around both surveillance and response. The Department of Health has chaired a UK WNV task force since the end of 2000. This is a multidisciplinary group of senior representatives from Agencies and Government Departments involved in human and animal health, entomology and academic departments. Activities include surveillance for WNV infections in humans, and in dead birds, mosquitoes and horses. All have been negative for WNV. A WNV contingency plan was produced in 2004, and this could be used as a generic plan for an effective and coordinated response in the event of the emergence of a new vector-borne zoonotic infection.     

A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH₄Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.     

Anthropometric and Micronutrient Status of School-Children in an Urban West Africa Setting: A Cross-Sectional Study in Dakar (Senegal)

Background

Urban areas in West Africa are not immune to undernutrition with recent urbanization and high food prices being important factors. School children often have a poor nutritional status, potentially affecting their health and schooling performance. Yet, generally school children do not benefit from nutrition programs. The objective of the study was to assess the anthropometric and micronutrient status of children from state schools in the Dakar area.

Methods

School children (n = 604) aged from 5 to 17 y (52.5% girls, 47.5% ≥10 y) were selected through a two-stage random cluster sample of children attending urban primary state schools in the Dakar area (30 schools × 20 children). The prevalence of stunting (height-for-age<−2 z-scores) and thinness (BMI-for-age<−2 z-scores, WHO 2006, and three grades of thinness corresponding to BMI of 18.5, 17.0 and 16.0 kg/m2 in adults) were calculated from weight and height. Hemoglobin, plasma concentrations of ferritin (FER), transferrin receptors (TfR), retinol binding protein (RBP), and zinc, and urinary iodine concentrations were measured. Correction factors were used for FER and RBP in subjects with inflammation determined with C-reactive protein and α1-acid-glycoprotein.

Results

4.9% of children were stunted, 18.4% were thin, 5.6% had severe thinness (BMI-for-age<−3 z-scores). Only one child had a BMI-for-age>2 z-scores. Prevalence of anemia, iron deficiency and iron deficiency anemia was 14.4%, 39.1% and 10.6% respectively. 3.0% had vitamin A deficiency, 35.9% a marginal vitamin A status, and 25.9% zinc deficiency. Urinary iodine was <50 µg/L in 7.3% of children and ≥200 µg/L in 22.3%. The prevalence of marginal vitamin A, zinc deficiency, high TfR was significantly higher in boys than in girls (P<0.05). Height-for-age and retinol were significantly lower in participants ≥10 y and <10 y respectively.

Conclusion

Undernutrition, especially thinness, iron and zinc deficiencies in school children in the Dakar area requires special targeted nutrition interventions.     

Minimum Incidence of Adult Invasive Pneumococcal Disease in Blantyre,Malawi an Urban African Setting: A Hospital Based Prospective Cohort Study

Invasive pneumococcal disease causes substantial morbidity and mortality in Africa. Evaluating population level indirect impact on adult disease of pneumococcal conjugate vaccine (PCV) programmes in infants requires baseline population incidence rates but these are often lacking in areas with limited disease surveillance. We used hospital based blood culture and cerebrospinal fluid surveillance to calculate minimal incidence of invasive pneumococcal disease in the adult (≥15 years old) population of Blantyre, a rapidly growing urban centre in southern Malawi, in the period preceding vaccine introduction. Invasive pneumococcal disease incidence in Blantyre district was high, mean 58.1 (95% confidence interval (CI): 53.7, 62.7) per 100,000 person years and peaking among 35 to 40 year olds at 108.8 (95%CI: 89.0, 131.7) mirroring the population age prevalence of HIV infection. For pneumococcal bacteraemia in urban Blantyre, mean incidence was 60.6 (95% CI: 55.2, 66.5) per 100,000 person years, peaking among 35 to 40 year olds at 114.8 (95%CI: 90.3, 143.9). We suspected that our surveillance may under-ascertain the true burden of disease, so we used location data from bacteraemic subjects and projected population estimates to calculate local sub-district incidence, then examined the impact of community level socio-demographic covariates as possible predictors of local sub-district incidence of pneumococcal and non-pneumococcal pathogenic bacteraemia. Geographic heterogeneity in incidence was marked with localised hotspots but ward level covariates apart from prison were not associated with pneumococcal bacteraemia incidence. Modelling suggests that the current sentinel surveillance system under-ascertains the true burden of disease. We outline a number of challenges to surveillance for pneumococcal disease in our low-resource setting. Subsequent surveillance in the vaccine era will have to account for geographic heterogeneity when evaluating population level indirect impact of PCV13 introduction to the childhood immunisation program.     

Microsampling of Fish Otoliths: A Comparison Between DM 2800 and Dremel in Stable Isotope Analysis

This paper describes and compares two microsampling methods, DM 2800 and Dremel, which were used in taking aragonite samples from otolith zones of cod, Gadus morhua, for stable isotope analysis. There were no significant differences between the two sampling methods both in carbon and oxygen isotope ratios (¹³C/¹²C, ¹⁸O/¹⁶O) and in annual and seasonal otolith zones. The possible finite effect with Dremel could be minimized or negligible by selecting otoliths of younger fishes. Thus, Dremel may be accepted as a convenient microsampling method in stable isotope analysis of otoliths.     

3种城市生境间蚂蚁群落组成的比较研究——以四川省南充市城区为例

作为重要的人为影响因素,城市化改变了自然生境的景观结构,并影响到动物群落的组成。蚁科Formicidae动物种类繁多,且易于捕获,是研究城市中不同土地利用类型对动物群落影响的理想指示生物。2018年5月,在四川省南充市城区中选择"公园与绿地""旱地"和"乔木林地"3种土地利用类型作为不同的生境类型,并通过陷阱法对蚂蚁群落组成进行调查。共采集到1 847只个体;其中,津岛铺道蚁Tetramorium tsushimae在"公园与绿地"中的个体数占比最高(73.80%),并明显高于"旱地"群落中占比最高的中华小家蚁Monomorium chinense(25.48%)和"乔木林地"群落中占比最高的宽结大头蚁Pheidole nodus(28.79%)。3种生境间,群落的蚂蚁个体数和物种丰富度间差异无统计学意义,但"公园与绿地"的Simpson多样性指数和Pielou均匀度指数均显著低于"旱地"和"乔木林地"的;"公园与绿地"(q=0.444)和"旱地"(q=0.500)与"乔木林地"间的群落组成均存在中度不相似,而"公园与绿地"与"旱地"间为中度相似(q=0.647)。因此,不同的城市土地...     

Detection of the West Nile Virus and its genetic typing in ixodid ticks (Parasitiformes: Ixodidae) in Tomsk City and its suburbs

Four tick species, Ixodes persulcatus, I. pavlovskyi, I. trianguliceps, and Dermacentor reticulatus, were found in Tomsk and its suburbs in 2006. The species I. pavlovskyi was found to be dominant in the localities situated in Tomsk City, and I. persulcatus was dominant in its suburbs. Viral RNA and viral antigen of the West Nile virus (WNV) were detected in the ticks I. pavlovskyi and I. persulcatus collected in the city and its suburbs by the RT PCR method and enzyme immunoassay with monoclonal antibodies against protein E of the WNV. Average rate of the WNV infected ticks varied from 5.2 up to 11.7% in different localities. Identification of the nucleotide sequence of the protein E gene fragment allowed classifying the cDNA obtained as genotype Ia of the WNV. The sequences are proved similar to the strain LEIV-Vlg99-27889-human of the WNV isolated in Volgograd. The obtained data showed that natural foci of the WNV virus can appear in the city and its suburbs probably involving two dominant tick species. The WNV infected imagoes, larvae, and nymphs of I. persulcatus and I. pavlovskyi were collected from small mammals, lizards, and birds. Therefore we presume that these hosts can be involved in the circulation and distribution of WNV on the territory of Tomsk Region.